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To determine the molecular basis, genotype - phenotype relationship, and genetic origin of Hemoglobin (Hb) Hekinan associated with several forms of α-thalassemia and other hemoglobinopathies for a better understanding of its diverse clinical phenotypes. Seventeen participants with suspected abnormal Hb were studied. Hb analysis was performed using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Mutational and α-haplotypic and structural analyses were conducted, and the effects of mutations on globin-chain stability were determined. All participants harbored Hb Hekinan II (HBA1:c.84 G>T) co-inherited with another α-globin gene anomaly. Three novel genotypes, (αα Hekinan /α CS α), (αα Hekinan /α CS α,β A /β E ), and (αα Hekinan /α CS α,β E /β E ), were characterized. Despite being co-inherited with both α- and β-Hb variants Hb Hekinan II led to minimal changes in erythrocyte parameters, suggesting a non-pathological nature. HPLC but not CE revealed a distinct small shoulder-like Hb pattern. Thai Hb Hekinan II was strongly associated with haplotype [+ - S + - - -] and the possibility of four different haplotypes, while two Burmese Hb Hekinan II were associated with haplotypes [± - S + - + -] and [± - S + - - -]. The novel genotypes identified provide a fresh perspective on Hb Hekinan II diversity. HPLC has superior identification capabilities for samples of Hb Hekinan II co-inherited with α-thalassemia. Thai and Burmese Hb Hekinan II have diverse origins.

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 β 0 -thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

Background and Aims: We aimed to evaluate the efficiency of identification and quantification of hemoglobin (Hb) Constant Spring (CS) and Hb Paksé by capillary electrophoresis (CE)., Materials and Methods: Blood samples collected from 2057 patients were used for identifying and quantifying Hb by CE. Molecular analysis of α- and β-thalassemia, Hb CS, and Hb Paksé was performed., Results: Hb CS and Hb Paksé were identified in 573 samples (27.86%) with diverse genotypes. Thirty-eight samples (6.6%) showed no Hb CS peak. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of Hb CS by CE were 93.37, 95.96, 89.92, 97.40, and 95.24%, respectively. The amount of Hb CS in those carrying Hb CS was 0.2-6.5% which showed an increasing trend according to the number of defective α-globin genes, in contrast to Hb A 2 levels, which decreased. Hb CS level ≥1.0% accurately excluded heterozygotes and that of ≥2.0% could identify homozygotes., Conclusion: CE has the high potential for identifying and quantifying Hb CS and Hb Paksé, especially in an area with a high prevalence of thalassemia. Hb CS levels can be used as a potential marker to distinguish the genotype of individuals carrying Hb CS., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

α-thalassemia is an inherited blood disorder that is most frequently found in Southeast Asian populations. In Thailand, molecular characterization can diagnose most patients with α-thalassemia; however, several atypical patients are also observed in routine analyses. Here, we characterized α-thalassemia mutations among 137 Hemoglobin H (Hb H) disease patients and three fetuses of Hb Bart's hydrops, a fatal clinical phenotype of α-thalassemia. Specifically, we performed multiplex ligation-dependent probe amplification (MLPA) followed by direct DNA sequencing. We noticed common genotypes in 129 patients and eight patients had rare Hb H disease caused by compound heterozygous α 0 -thalassemia (-- CR or -- SA deletion) with α + -thalassemia (-α 3.7 /-α 4.2 /α Constant Spring α). Furthermore, two affected fetuses had the -- SA /-- SEA and one had the -- CR /-- SEA genotypes. Next, we developed and validated a new multiplex gap-PCR and applied this method to 844 subjects with microcytic red blood cells (RBCs) from various parts of Thailand. The frequency of heterozygous α 0 -thalassemia was dominated by -- SEA 363/844 (43%), followed by -- THAI 3/844 (0.4%), -- SA 2/844 (0.2%), and -- CR 2/844 (0.2%) mutations. These findings suggest that aforementioned four mutations should be routinely applied to increase the effectiveness of diagnosis and genetic counseling in this region., (© 2023. The Author(s).)

Background: An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important., Objective: This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α + -thalassemia phenotype in carriers with and without other hemoglobinopathies., Materials and Methods: Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism were developed., Results: Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA 2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α 3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of α Doi-Saket with β E globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis., Conclusions: Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α 3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management.

Background: Low HbF expression in HbE-β + -thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the β - and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-β + -thalassemia from HbE heterozygotes have been investigated., Participants and Methods: A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize β- and α-globin genes. DNA polymorphism at position -158 nt 5' to G γ-globin was performed by Xmn I restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA 2 , HbE, and HbF levels for the differentiation of HbE-β + -thalassemia from HbE heterozygotes were determined., Results: Five β + -thalassemia mutations trans to β E -gene (β -87(C>A) , β -31(A>G) , β -28(A>G) , β 19(A>G) , and β 126(T>G) ) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000-1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden's index for differentiating HbE-β + -thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-β + -thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α 2 -globin genes were identified. The genotype and allele of the polymorphism at -158 nt 5' to G γ-globin was found to be negatively associated with HbF expression., Conclusions: HbE-β + -thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-β + -thalassemia diagnosis.

The interactions of δ-globin variants with α- and β-thalassemia or other hemoglobinopathies cause complex thalassemic syndromes and potential diagnostic problems. Understanding the molecular basis and phenotypic expression is crucial. Four unrelated Thai subjects with second hemoglobin (Hb) A2 fractions were studied. A standard automated cell counter was used to acquire initial hematological data. Hb analysis was carried out by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assays. Globin gene mutations and haplotype were identified by appropriate DNA analysis. An allele-specific polymerase chain reaction method was developed to provide a simple molecular diagnostic test. Hb analysis revealed a Hb A2 variant in all cases. DNA analysis of the δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu > Lys] variant in combination with Hb E in three cases. Analysis of the remaining case identified a novel δ-Hb variant, namely Hb A2-Mae Phrik [δ52(D3)GAT > GGT; Asp > Gly], found in association with Hb E and α+-thalassemia, indicative of the as yet undescribed combination of triple heterozygosity of globin gene defects. An allele-specific PCR-based assay was successfully developed to identify this variant. The β-haplotype of the Hb A2 Mae-Phrik allele was strongly associated with haplotype [+ − − − − ± +]. This study advanced our understanding of the phenotypic expression of known and novel δ-Hb variants coinherited with other globin gene defects, routinely causing problems with diagnosis. Therefore, knowledge and recognition of this Hb variant and molecular assessments are crucial to improving diagnosis.

A misdiagnosis of β-thalassemia carrier in samples with Hb Tak and HbD-Punjab, the β-variants, can be a cause of inappropriate genetic counseling thus having a new case of β-thalassemia major. A capillary electrophoresis (CE) is very efficient in separating and quantifying HbA 2 . In this study, HbA 2 levels of samples which were doubted for compound heterozygous Hb Tak/β-thalassemia or heterozygous HbD-Punjab/β-thalassemia were measured and compared between CE and high performance liquid chromatography (HPLC). The molecular confirmation for Hb Tak, HbD-Punjab and β-thalassemia codons 17 (A > T), 41/42 (-TCTT), 71/72 (+A) and IVSI-nt1 (G > T) mutations and 3.4 kb deletion were also performed. Based on DNA analysis, 3 cases were diagnosed as compound heterozygous Hb Tak/β-thalassemia and one for HbD-Punjab/β-thalassemia. The elevated HbA 2 levels were found in all 4 samples with rages of 4.6-7.3% on CE while those were not found on HPLC. Thus, the elevated HbA 2 measured by CE can be used as a screening parameter for differentiating the homozygote of Hb Tak and HbD-Punjab from the compound heterozygote of these hemoglobinopathies and β-thalassemia., Competing Interests: Compliance with Ethical StandardsThe authors report no conflicts of interest.This study was approved by the Institutional Review Board (IRB) of the University of Phayao, Phayao, Thailand (Ethical Approval Number HE5403010001).The informed consent was obtained from the subjects included in the study.

Hemoglobin (Hb) New York [β113 (G15) Val→Glu, GTG>GAG] is a very rare β-chain variant found in Thailand. This variant is often missed by routine laboratory testing because Hb New York and Hb A have the identical retention time on high performance liquid chromatography. We reported here for the first time that the detection of Hb New York in a Thai woman by using capillary electrophoresis (CE). A peak of Hb New York located ahead of Hb A at the electrophoretic zone 11 with a level of 42.8 %. The DNA sequencing revealed the GTG>GAG mutation at codon 113 for Hb New York on one allele of β-globin gene. Therefore, the CE has a high efficiency to prevent the misinterpretation of hemoglobin analysis in patients who are heterozygote of this variant., Competing Interests: The authors report no conflicts of interest. Informed consent This study was approved by the Ethics Committee of the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand. The informed consent was obtained from all individual participants included in the study.

Hemoglobin (Hb) J-Buda [α61(E10)Lys → Asn, AAG > AAT] is a very rare α-chain variant found in South-East Asia. We analyzed hematological parameters and provided a rapid molecular analysis method for detection of this hemoglobinopathy in two Thai women who had severe microcytic anemia with Hb and MCV <70 g/L and 80 fL, respectively. The HPLC revealed an abnormal Hb peak eluted ahead of HbA at retention time of 1.91-1.98 min. On CE, the abnormal Hb peak was observed at the electrophoretic zone 12 which corresponded to Hb Bart's. The DNA sequencing revealed the AAG → AAT mutation at codon 61 for Hb J-Buda on one allele of the α1-globin gene. The developed Allele-specific PCR (ASPCR) showed the 455 bp amplified fragment from Hb J-Buda allele. Thus, understanding of hematological characterizations and the developed ASPCR for diagnosis of Hb J-Buda are essential for genetic counseling of this hemoglobinopathy.

We report a hitherto undescribed interaction of a deletional (deltabeta) degrees -thalassemia and a deletional hereditary persistence of fetal hemoglobin (HPFH) in an adult Thai individual. He was a 40-year-old Thai male who had the following hematologic data: Hb 13.9 g/dL, Hct 43.8%, MCV 78.0 fL, MCH 24.7 pg, MCHC 31.6 g/dL, and RDW 17.1%. Hemoglobin analysis revealed 97% Hb F with Ggamma-globin chain predominant. Globin gene analyses demonstrated that he carried the GgammaAgamma(deltabeta) degrees -thalassemia deletion in trans to the HPFH-6. Hematologic data of the patient were compared to those of the heterozygotes for these high-Hb F determinants found in his parents and an unrelated Thai patient with a compound HPFH-6/deletion-inversion Ggamma(Agammadeltabeta) degrees -thalassemia previously described., ((c) 2005 Wiley-Liss, Inc.)

Background and Objectives: Hereditary persistence of fetal hemoglobin (HPFH) and deltabeta-thalassemia are heterogeneous disorders characterized by increased levels of Hb F in adult life. The distinction between these two conditions is not always possible from routine hematologic analyses. This study investigated the hematologic and molecular characteristics of high HbF determinants in Thailand, and describes a rapid DNA-based assay to facilitate diagnosis in a routine laboratory., Design and Methods: A multiplex allele-specific polymerase chain reaction (PCR) system for rapid detection of three common DNA deletions causing (deltabeta)0-thalassemia and HPFH in South-east Asians was developed and used to examine the molecular basis for the high Hb F phenotypes in 273 unrelated Thai individuals. Hematologic data were recorded and correlated to the molecular findings., Results: The multiplex PCR system was validated and results were completely concordant with those of other established methods. DNA analysis identified GgAg(deltabeta)0-thalassemia in 148 cases (54.2%), deletional HPFH-6 in 83 (30.4 %) and the deletion-inversion Ggamma(Agammadeltabeta)0-thalassemia in 22 (8.1 %) cases, while another 20 (7.3 %) subjects remained uncharacterized. Genotype-phenotype relationships are discussed., Interpretation and Conclusions: These data emphasize the high frequencies of deltabeta-thalassemia and HPFH in Thailand and the need for differential diagnostic methods since the hematologic parameters associated with the conditions are very similar and overlap. The multiplex allele-specific PCR approach should prove useful in complementing routine Hb analysis for the differential diagnosis of these three common causes of high Hb F determinants and should facilitate a program of hemoglobinopathy screening in the region.

Background and Objectives: This study aimed to describe hematologic and molecular characterization of the interaction of hemoglobin (Hb) E and several forms of alpha-thalassemia causing complex thalassemia syndromes in two Cambodian families as well as to establish a rapid polymerase chain reaction (PCR) assay for simultaneous detection of Hb Constant Spring (CS) and Hb Pakse' (PS)., Design and Methods: Using PCR and DNA sequencing, the alpha- and beta-globin genotypes were examined. Clinical and hematologic data were assessed. A multiplex asymmetric allele-specific PCR for differential diagnosis of HbCS and HbPS was developed and validated., Results: Eight genotypes including heterozygous HbCS, heterozygous HbPS, double heterozygous HbE/HbPS, double heterozygous HbE/alpha-thalassemia 2, triple heterozygous HbE/alpha-thalassemia /HbPS, homozygous HbE/alpha-thalassemia 2, compound alpha-thalassemia 2/HbCS and a hitherto undescribed compound HbCS/HbPS were found in these two families. Genotype-phenotype relationships are discussed and successful application of a multiplex PCR system for differential diagnosis of HbCS and HbPS is described., Interpretation and Conclusions: The interaction of several globin gene abnormalities in Cambodian families emphasizes the high frequencies of thalassemia and hemoglobinopathies. Identification of HbPS suggests that this mutation might be common and underestimated among South-east Asian populations. A simplified PCR assay for simultaneous detection of HbCS and HbPS would facilitate characterization of these genotypes in both the clinical setting and population screening programs in the region.