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Background: Excessive expression of hemoglobin F (HbF) is a characteristic feature and important diagnostic marker of β 0 -thalassemia/HbE disease. However, some patients may exhibit low-HbF levels, leading to misdiagnosis and precluding genetic counseling. The genetic factors influencing these differences in HbF expression in this atypical disease are not completely understood., Aims: To investigate determinants contributing to the non-elevation of HbF expression in β 0 -thalassemia/HbE disease., Methods: We studied 231 patients with β 0 -thalassemia/HbE confirmed by DNA analysis; classified them into the low-HbF (n = 62) and high-HbF (n = 169) groups; analyzed hematological parameters and hemoglobin levels in both groups; and characterized mutations in β- and α-globin genes and genetic variants in γ-globin promoters., Results: Both groups showed similar rates of type β 0 -thalassemia mutations but significantly different proportions of α-globin mutations: approximately 88.7% (95% confidence interval [CI] = 66.8-115.5) and 39.1% (95% CI = 30.2-49.7) in the low- and high-HbF groups, respectively. The results revealed single-nucleotide polymorphisms (SNPs) at -158 (C>T) in the G γ-globin promoters and novel SNPs at the 5' untranslated region position 25 (G>A) in A γ-globin promoters. The distribution of CC genotypes of the G γ-globin promoter in the low-HbF group was significantly higher than that in the high-HbF group., Conclusions: Cases with HbE predominance with low-HbF levels and undetectable HbA may not be as conclusive as those with homozygous HbE until DNA analysis is performed. Concomitant inheritance of α-thalassemia is an important inherent factor modifying HbF expression in a typical β 0 -thalassemia/HbE, and SNPs with the CC genotype in the G γ-globin promoter may indicate unelevated HbF expression in patients with this disease., (Copyright © 2024 Instituto Mexicano del Seguro Social (IMSS). Published by Elsevier Inc. All rights reserved.)

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.

Objectives: Accurate quantification of hemoglobin (Hb) A 2 is vital for diagnosing β-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA 2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE)., Methods: We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA 2 cutoff value for β-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the β- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing., Results: HbA 2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias, -0.33; r, 0.991), β-thalassemia (bias, -0.27; r, 0.927), and non-β-thalassemia carriers (bias, -0.36; r, 0.928). An HbA 2 cutoff value of ≥4.0 % for β-thalassemia screening achieved 100 % sensitivity and 99.6 % specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3 , 99.8, 97.3, 99.8, and 99.7 %, respectively. We also identified a rare β-Hb variant, Hb La Desirade [ HBB :c.389C>T], associated with β-thalassemia and co-inherited with a single α-globin gene., Conclusions: The Premier Resolution HPLC is a reliable and accurate method for routine β-thalassemia carrier screening, aligning with existing CE methods., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

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.

Background: Delta-chain (δ-chain) variants are a group of rare hemoglobin (Hb) variants resulting from mutations within the δ-globin gene. Although quantification of Hb A2 levels is a useful screening tool for the beta-thalassemia trait, the coinheritance of a δ-globin gene mutation can lead to misinterpretation of diagnostic results. Objective: To identify an unreported Hb A2 variant in Thailand and to develop a high resolution melting (HRM) curve assay for the four δ-globin chain variants found in the Thai population. Materials and Methods: Allele-specific polymerase chain reaction (ASPCR) was used to analyze a total of 18 DNA samples for Hb variants comprising 10 wild-type controls, 4 Hb A2-Melbourne, 1 Hb A2-Lampang, 2 Hb A2-Kiriwong, and an unknown variant via HRM assays. Results: The unreported Hb A2 variant in Thailand was found to be Hb A2-Walsgrave resulting from δ-globin gene mutation at codon 52 (GAT>CAT). This was also confirmed using ASPCR. In addition, we demonstrated that the HRM curve profile for Hb A2-Melbourne, Hb A2-Lampang, Hb A2-Walsgrave, and Hb A2-Kiriwong could be identified so as to distinguish the mutant alleles from one another and from wild-type alleles. Conclusion: This HRM assay detected both known and unknown mutations with simultaneous differentiation between heterozygous and homozygous alleles on a polymerase chain reaction fragment spanning four of the δ-globin variants found in Thailand. This assay may help to support the prevention and control of thalassemias and hemoglobinopathies in Thailand.

Interaction of structural hemoglobin (Hb) variants with α- or β-globin defects are occasional in Southeast Asia. Herein we provide the first description of Hb Athens-Georgia (Hb A-Ga) in association with deletional Hb H disease, a novel combination previously undescribed in the population. Hematological, Hb and DNA analysis, and β-globin haplotype analyses were performed in seven participants from one ethnic Thai family. Hemoglobin analysis by capillary electrophoresis revealed an abnormal Hb fraction in the proband, his father and grandmother (I-2). DNA sequencing revealed that the G > A substitution at codon 40 of the β-globin gene was identical to the Hb A-Ga ( HBB :c.122G > A). Interestingly, α-thal-1 (SEA deletion) and α-thal-2 (-α 3.7 deletion) were identified in the proband resulting in Hb H disease, while α-thal-1 was identified in the father, and no α-thal was observed in I-2. Hematological analysis indicated that the proband (β A-Ga /β A , - SEA /-α 3.7 ) had moderate anemia and was markedly hypochromic with microcytic red blood cells (RBCs). The father (β A-Ga /β A , - SEA /αα) presented mild microcytic anemia, while normal hematology was observed in the I-2 who was heterozygous for Hb Athens-Georgia (β A-Ga /β A , αα/αα). The relative level of Hb A-Ga was distinctly reduced according to the degree of α-globin defects. The developed allele-specific PCR method can successfully be used for confirmation of Hb A-Ga. The Thai Hb A-Ga allele associated with a β-haplotype [+ - - - - - +]. These findings were in accordance with the previous conclusion that this variant is a non-pathological β-Hb variant.

Hb E [β26(B8)Glu→Lys, G AG> A AG, HBB : c.79G>A] is an inherited thalassemic β-globin variant that favors the Hb E-β-thalassemia (β-thal) syndrome when interacting with the β-thal gene. However, hemoglobin (Hb) variants carrying Hb E in combination with another variant on the same β gene are rare. We recently studied a 29-year-old pregnant woman, initially diagnosed as a β-thal carrier. Hemoglobin and DNA analysis were performed by high performance liquid chromatography (HPLC) and DNA sequencing. Hematological data revealed no anemia or altered red blood cell (RBC) parameters. Hemoglobin HPLC showed Hb A and Hb A 2 but no Hb E or abnormal Hb peaks, with a markedly elevated Hb A 2 level (6.4%) reaching the accepted range (4.0-10.0%) for β-thal trait. DNA analysis identified a G AG> A AG transition at codon 26 of the β-globin gene that is responsible for Hb E, and an AA G >AA C mutation at codon 65 in cis on the β-globin chain resulting in a lysine to asparagine substitution. These two mutations led to the formation of a novel variant, namely Hb E-Myanmar, β26(B8)Glu→Lys and β65(E9)Lys→Asn, HBB : c.[79G>A;198G>C]. Moreover, a heterozygous α-thalassemia-2 (α-thal-2) [-α 3.7 (rightward)] deletion was also observed. Hb E-Myanmar is a doubly substituted β-globin variant, which has not been previously described. This variant did not have any clinical or hematological abnormalities, and the genetic mechanism resulting in this variant is discussed. The new simultaneous allele-specific polymerase chain reaction (ASPCR) was developed for rapid detection of these two mutations within the same β-globin chain.

Hb H diseases with the clinical features of thalassemia are found in many parts of the world, including Southeast Asia and southern China. There are limitations in molecular data from the population of Thailand, which includes multiple ethnic groups. Here, we characterized the molecular basis of the disease among a large cohort from this region. A total of 479 unrelated Thai patients with Hb H disease were studied. Mutations of the α-globin gene were characterized by conventional gap-PCR and rare genotypes were identified by MLPA analysis and direct DNA sequencing. The molecular characterization showed five common Hb H genotypes (472/479; 98.54%), including three deletional types (- SEA /-α 3.7 ; n  = 312), (- SEA /-α 4.2 ; n  = 26), (- THAI /-α 3.7 ; n  = 1) and two non-deletional types (- SEA /α CS α; n  = 131), (- SEA /α Paksé α; n  = 2). Herein, we firstly report a rare genotype of Hb H disease with (- SA /-α 3.7 ; n  = 1) that has not been documented in Thailand, and rare genotypes related to (- SEA /-α 16.6 ; n  = 1), and (- SEA /α QS α; n  = 3) as well. The remaining two cases could not be characterized. The hematological parameters demonstrated that the clinical phenotype of non-deletional Hb H diseases is more severe than the deletional type of α + -thalassemia. The molecular spectrum of α-thalassemia is useful for prevention and thalassemia control and genetic counseling for couples at risk in this region.

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 A 2 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.

Elevated Hb A 2 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 A 2 levels are below 4.0%. In addition, coinheritance of α-thalassemia (α-thal) and β-thal might affect Hb A 2 levels. Therefore, the aim of this study was to investigate the mutations of β- and α-globin genes in individuals with borderline Hb A 2 levels in Thailand. Three hundred samples from individuals with Hb A 2 levels of 3.5-3.9% were collected for molecular diagnosis of β-globin gene mutations. In addition, the α 0 -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 α 0 -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 A 2 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 A 2 levels, leading to a misdiagnosis of β-thal in analysis programs.

The clinical consequences of many abnormal hemoglobins (Hbs) interacting with α- or β-thalassemia (α- or β-thal) or other hemoglobinopathies have not been described. We evaluated a 75-year-old Thai woman and her 45-year-old daughter. Hematological data was obtained on an automated cell counter. Hemoglobin (Hb) analysis was carried out using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) assays. Mutations and globin haplotypes were identified by appropriated DNA techniques. The proband presented with moderate anemia and inclusion bodies in most of the red blood cells (RBCs), while altered RBC parameters were absent in her daughter. Hemoglobin analysis showed an abnormal Hb peak only in the proband. DNA analysis identified a G>T substitution at codon 31 of the α1-globin gene, corresponding to Hb Prato [α31(B12)Arg→Ser ( HBA1 or HBA2 c.96G>T or C)] in both subjects. The α-thal-1 [- - SEA (Southeast Asian)] deletion was also identified in the proband, but not in her daughter. These mutations could be identified using newly developed allele-specific polymerase chain reaction (ASPCR) assays. The α haplotypic analysis demonstrated the Thai Hb Prato allele was associated with haplotype [+ - S + - + -] [the S represents the inter ζ hypervariable region (HVR)]. The combination of the unstable Hb Prato with α-thal-1 result in α-thal intermedia (α-TI) phenotypes. A simple DNA method is essential for detection, and a haplotypic α-globin gene cluster are presented.

In this study, Hb A 2 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), -α 3.7 (rightward) and -α 4.2 (leftward)] deletions, and Hb Constant Spring (Hb CS; HBA2 : c.427T>C), Hb A 2 -Melbourne ( HBD : c.130G>A), Hb A 2 ' ( HBD : c.49G>C), Hb A 2 -Lampang ( HBD : c.142G>A). β 0 -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 A 2 variant peak in zone 1 of the capillary electrophoresis (CE) electropherogram. Results showed that 20 patients (87.0%) carried Hb A 2 -Melbourne with seven different genotypes for α- and β-thal, two (8.7%) carried Hb A 2 ' and one (4.3%) carried Hb A 2 -Lampang. All three samples doubly heterozygous for Hb A 2 -Melbourne/β 0 -thal had Hb A 2 levels lower than 4.0%, while summation of Hb A 2 and Hb A 2 -Melbourne ranged from 4.9-5.3%, reaching the accepted range (4.0-10.0%) for β-thal trait. Hb A 2 -Melbourne is the most common δ-globin variant in the Thai population. Hb A 2 variant and Hb A 2 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 A 2 -Melbourne, Hb A 2 ' and Hb A 2 -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 A 2 variants.

The capillary electrophoresis (CE) system allows the quantification of Hb Bart's (γ4) and Hb H (β4) that is used for screening of Hb H disease. However, Hb Bart's hydrops fetalis and Hb H are not always codetected in patients with Hb H disease. In this study, 35 samples were analyzed for the α 0 -thalassemia (α 0 -thal) [- - SEA (Southeast Asian) and - - THAI (Thailand)] deletions and the α + -thal [-α 3.7 (rightward) and -α 4.2 (leftward)] type deletions using real time-polymerase chain reaction (real time-PCR) with SYBR Green1 and high-resolution melting (HRM) analysis and conventional gap-PCR techniques, respectively. Results showed that 28 of 29 (96.6%) samples with the Hb A 2 -Hb H phenotype on CE electrophoregrams presented the genotype of - - SEA /-α 3.7 , while the - - SEA /-α 4.2 made up the remainder. The - - SEA /-α 3.7 genotype was also found in all six samples (100.0%) with Hb A 2 -Hb Bart's on CE electrophoregrams. Thus, for genetic counseling, prevention and control programs of Hb Bart's hydrops fetalis and Hb H disease, α-thal genotype analysis is required.

We report the molecular and hematological identifications of a Hb A 2 variant [coinheritance of Hb A 2 -Melbourne ( HBD : c.130G>A) and Hb E ( HBB : c.79G>A)] found for the first time in the Lao People's Democratic Republic (PDR). The subject was a 29-year-old pregnant Laotian woman who was a foreign worker in Thailand and was diagnosed with thalassemia and hemoglobinopathies. Capillary electrophoresis (CE) demonstrated 1.6% of Hb A 2 , with a minor unknown peak at the initial Z1 zone (1.7%). Identification of abnormal hemoglobin (Hb) using direct DNA sequencing showed a genetic defect causing a δ-globin gene missense mutation at codon 43 ( G AG> A AG) causing a glutamic acid to lysine substitution corresponding to Hb A 2 -Melbourne. The origin of Hb A 2 -Melbourne in Lao PDR may be similar to a case found in Thailand with the [+ - - - - + +] haplotype. We developed a method that could clearly detect Hb A 2 -Melbourne and Hb A 2 -Lampang ( HBD : c.142G>A) mutations in a single tube using high resolution melt (HRM) analysis. The HRM analysis is a more effective method for rapid detection than conventional polymerase chain reaction (PCR), as there is no need for a post-PCR step, and no exposure to ethidium bromide. This new method would be a useful addition for the first investigation of a suspected Hb A 2 variant in the routine molecular setting.

Hb A' 2 (or Hb B 2 ) ( HBD : c.49G>C) is the most frequent δ chain variant that has been described in Africa but not in Thailand. We report here a 10-month-old Thai infant with compound heterozygosity for β 0 codon 17 (A>T; HBB : c.52A>T) and β + IVS II-654 (C>T; HBB : c.316-197C>T). Under diagnosed β-thalassemia (β-thal) in her father, who carries Hb A' 2 and a heterozygous β 0 codon 17 mutation, and the mother, who carries a heterozygous β + IVS II-654 mutation, was noted. Although Hb A' 2 does not cause any problems, heterozygosity for Hb A' 2 can lead to under diagnosis of β-thal in Hb A' 2 samples. This case highlights the importance of Hb A' 2 in prenatal diagnosis (PND). Thus, molecular analysis for β-thal mutations should be carried out when a small peak presents at the retention time (RT) of 4.71 min. on high performance liquid chromatography (HPLC) and the summation level of this peak and Hb A 2 was equal or higher than 4.0%.

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.

Hb Q-Thailand [α74(EF3)Asp→His (α1), GAC>CAC, HBA1: c.223G>C] is an abnormal hemoglobin (Hb) frequently found in Thailand and Southeast Asian countries. The association of the α Q-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report how to diagnose the coinheritance of Hb Q-Thailand with β-thalassemia (β-thal)/Hb E disease in four Thai samples from high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) testing results. Understanding of the HPLC chromatogram and CE electropherogram patterns of this complex mutation is important for interpretation of testing results and providing genetic counseling.

We report the hematological parameters and provide a rapid molecular analysis method for detection of Hb Wiangpapao [α44(CE2)Pro→Ser, CCG>TCG; HBA1: c.133C>T], a new α-globin variant found in a pregnant Thai woman. Her red cell indices were measured by an automated blood counter. The results were: red blood cell (RBC) count 4.03 × 10 12 /L, Hb 13.1 (g/dL), packed cell volume (PCV) 0.39 L/L, mean corpuscular volume (MCV) 97.0 fL, mean corpuscular hemoglobin (Hb) (MCH) 32.5 pg, mean corpuscular Hb concentration (MCHC) 33.4 g/dL, and RBC distribution width (RDW) 9.4%. The Hb typing by high performance liquid chromatography (HPLC) showed 13.6% abnormal Hb at a retention time of 2.20 min. that was difficult to distinguish from Hb A. On the capillary electrophoresis (CE) electropherogram, this hemoglobinopathy peak did not separate from the Hb A peak. DNA sequencing showed a C>T transition at the first position of codon 44 (CCG>TCG) of the α1-globin gene that led to a substitution of proline for serine. This mutation has not been recorded in the public databases. Therefore, we named it Hb Wiangpapao as it was first discovered in the Wiangpapao District, Chiang Rai, Thailand. The multiplex allele-specific polymerase chain reaction (ASPCR) for detection of Hb Wiangpapao was developed and revealed a 510 bp specifically amplified fragment. The better understanding of hematological characterizations and the newly developed multiplex ASPCR for diagnosis of Hb Wiangpapao are useful for genetic counseling and family education.

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.

Hb Agenogi [β90(F6)Glu→Lys (GAG>AAG) HBB: c.271G>A)] is a very rare β-globin chain variant. We report for the first time this hemoglobinopathy in a pregnant 20-year-old Thai woman. She was seen by an obstetrician at her 14th week of gestation. She was pale and had an inflammatory lesion of her lower left leg. The hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) and low pressure liquid chromatography (LPLC) showed a peak of abnormal Hb at the C window. On capillary electrophoresis (CE), the abnormal Hb peak was observed at electrophoretic zone 4 that corresponded to the Hb E (HBB: c.79G>A) peak. Direct DNA sequencing revealed a GAG>AAG mutation at codon 90 of the β-globin gene. Thus, even though Hb Agenogi is very rare, it can be found in Thai people. The knowledge and understanding of this hemoglobinopathy will be used to assist in diagnosis, management and counseling for patients.

Background: There are limited data on hemoglobin (Hb) variants among peoples of northern Thailand. Hence, we determined the prevalence of Hb variants among a large cohort from this region., Methods: A study was done on 23,914 subjects recruited from eight provinces during June 2012-January 2014. Hb was analyzed by high performance liquid chromatography (HPLC) and capillary electrophoresis, and corresponding mutations were identified by polymerase chain reaction., Results: Among 23,914 subjects examined, 211 (0.88%) were found to carry 14 different Hb variants. Five α-globin chain variants were identified: Hb Q-Thailand (n = 40; 19.0%), Hb Hekinan (n = 8, 3.8%), Hb Siam (n = 2, 0.9%), Hb Beijing (n = 1, 0.5%), and Hb Kawachi (n = 1, 0.5%), not previously described in the Thai population. Seven β-globin variants, including Hb Hope, Hb Tak, Hb S, Hb J-Bangkok, Hb G-Makassar, Hb C, and Hb Korle-Bu, were found in 115 (54.5%), 30 (14.2%), 3 (1.4%), 3 (1.4%), 1 (0.5%), 1 (0.5%), and 1 (0.5%) subjects, respectively. The remaining five subjects (2.4%) were carriers of two different δ-globin chain variants. A different spectrum and frequencies of Hb variants were noted compared to other geographical areas. Haplotype analysis demonstrated multiple origins for Hbs Hope and Tak and confirmed a non-African origin of Hb C. Several genetic interactions between these variants with other hemoglobinopathies were encountered. Associated hematological phenotypes and novel Hb derivatives formed were presented., Conclusions: The prevalence and molecular heterogeneities of the Hb variants found in this large cohort of the northern Thai people's should prove useful in developing a screening program, and for the performance of additional population genetics studies of hemoglobinopathy in the region.

Background: We reported molecular and hematological characteristics of δ-globin chain variants and addressed diagnostic consideration of complex hemoglobinopathies caused by their interactions with α- and β-thalassemias., Methods: Study was done on four unrelated Thai subjects with second Hb A2 fractions. Hb analysis was carried out using automated HPLC and capillary electrophoresis. Mutations were identified by DNA analysis. Novel diagnostic methods based on PCR-RFLP and allele specific PCR were developed., Results: Hb analysis revealed Hb A2 variant in all cases. DNA analysis of δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu→Lys] in combination with α(+)-thalassemia, α(0)-thalassemia and β(0)-thalassemia in the first three cases, respectively. Analysis of the remaining case identified a novel δ-Hb variant namely the Hb A2-Lampang [δ47(CD6)GAT→AAT; Asp→Asn] found in association with Hb E and α(+)-thalassemia. These mutations could be identified using PCR-RFLP and allele specific PCR assays developed., Conclusions: It is necessary to recognize the Hb A2 variant and to combine the amounts of Hb A2 and Hb A2-variant for a total Hb A2 value to make better diagnostic of these complex syndromes. Co-inheritance of these multiple globin gene defects could lead to complex hemoglobinopathies requiring comprehensive Hb and molecular assessments., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report the molecular and hematological feature of a Thai woman who had clinical diagnosis of β-thalassemia intermedia (β-TI). Hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified Hb A (64.4%), Hb F (12.3%) and Hb A2/E (15.9%) with small peaks of Hb Bart's (γ4) and Hb H (β4). She was initially diagnosed as EA Bart's disease, which occurs from combination of Hb H disease and Hb E (HBB: c.79G > A) trait. However, the Hb analysis using capillary electrophoresis (CE) demonstrated no Hb E, 68.5% Hb A, 15.5% Hb F and 16.0% Hb A2. DNA analysis showed a compound heterozygosity for (β(+)) -31 (A > G) (HBB: c.-81A > G) and (β(0)) codon 17 (A > T) (HBB: c.52A > T) mutations and deletional Hb H (- -(SEA)/-α(3.7)). Thus, she was finally diagnosed with a combination of Hb H disease and compound heterozygosity of β(+)/β(0)-thalassemia (β(+)/β(0)-thal). The β-globin mutations could affect not only hematological parameters but also elevate the Hb A2 levels. These effects could not be ameliorated by the coinheritance of Hb H disease. Therefore, a better understanding of the effects of this combination on hematological analysis data will be useful for providing accurate diagnosis, genetic counseling, prevention and control programs of β-thalassemia major (β-TM).

Background: There have been no reports for the frequency of Hb Q-Thailand [alpha 74(EF3)Asp --> His, GAC > CAC] and its combinations either with other forms of thalassemia or hemoglobinopathies in Northern Thailand. The aims of this study were to search for Hb Q-Thailand and its combinations in Northern Thai population and to analyze fractions of hemoglobin in Hb Q-Thailand and its combinations on high performance liquid chromatography (HPLC) chromatograms and/or capillary electrophoresis (CE) electrophoregrams., Methods: Blood samples from public and private hospitals in 7 northern provinces of Thailand were analyzed for thalassemia and hemoglobinopathy diagnoses using HPLC and/or CE and DNA analysis techniques at the Thalassemia Laboratory, Associated Medical Sciences Clinical Service Center, Chiang Mai, Thailand., Results: Hb Q-Thailand was found in 13 of 13,596 (0.10%) samples; 6 were heterozygous Hb Q-Thailand, 4 were compound Hb Q-Thailand/alpha-thalassemia-1 Southeast Asian (SEA) type deletion and 3 with combinations of Hb Q-Thailand/beta(0)-thalassemia, Hb Q-Thailand/Hb E and Hb Q-Thailand/Hb E/alpha-thalassemia-1 SEA type deletion. The fractions of hemoglobin on HPLC chromatograms and CE electrophoregrams were observed based on types of combinations., Conclusions: Hb Q-Thailand and its combinations could be found in northern Thai population with the frequency of 0.10%. Thus, the better understanding of HPLC chromatogram and/or CE electrophoregram patterns of Hb Q-Thailand and its combination is essential for diagnosis and genetic counseling of thalassemia and hemoglobinopathies in this area.

Background: Hemoglobin (Hb) A(2) is artifactually elevated in cases of heterozygous Hb Hope when measured by capillary electrophoresis (CE). However, there is no report of HbA(2) levels and capillary electrophoregrams for associations of heterozygote of Hb Hope with α-thalassemia nor β-thalassemia., Methods: Levels of HbA(0), HbA(2) and Hb Hope in 16 heterozygous Hb Hope, 3 Hb Hope/α-thalassemia-1 SEA type deletion and 2 Hb Hope/β(0)-thalassemia were measured by CE. Electrophoregram and the levels of those were compared within these three groups., Results: Artifactually elevated HbA(2) levels (≥4%) were found in both groups of heterozygous Hb Hope and Hb Hope/α-thalassemia-1 SEA type deletion. Manual corrections were performed by adjusting baselines, and results showed that means of HbA(2) in both groups decreased from 4.47% and 4.03% to 1.93% and 1.77%, respectively. The highest levels of HbA(2) and Hb Hope were observed in samples with Hb Hope/β(0)-thalassemia. Moreover, HbA(0) was not observed in these cases., Conclusions: The elevation of HbA(2) in patients with heterozygous Hb Hope and with Hb Hope/α-thalassemia-1 SEA type deletion measured by CE leads to incorrect β-thalassemia trait diagnosis. However, using CE electrophoregram together with levels of HbA(0), HbA(2) and Hb Hope would be a more accurate and precise method for diagnosis of Hb Hope/β(0)-thalassemia.

The incidence of Hb Paksé (codon 142, TAA>TAT, α2) might have been underestimated due to misidentifying some cases as Hb Constant Spring (Hb CS, codon 142, TAA>CAA, α2) since both abnormal hemoglobins (Hbs) migrate to the same position on Hb electrophoresis or chromatography. Multiplex asymmetric allele-specific polymerase chain reaction (PCR) for identification of Hb CS and Hb Paksé, and a real-time PCR (ReTi-PCR) with SYBR Green1 high resolution melting (HRM) analysis, for detection of the α-thalassemia-1 (α-thal-1) Southeast Asian (- -(SEA)/) type deletion, were performed on 114 blood samples collected from subjects who lived in northern Thailand. These samples were previously identified as carrying Hb CS by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC). Five out of 114 (4.4%) samples were found to carry Hb Paksé with four different genotypes including Hb Paksé trait, compound Hb CS/Hb Paksé, Hb H-Hb Paksé disease and Hb H-Hb Paksé-Hb E disease. These results suggested that Hb Paksé and its various combinations can be misidentified as Hb CS. Although the clinical symptoms of Hb Paksé and Hb CS are similar, to prevent erroneous epidemiological data on Hb CS as well as underestimating the prevalence of Hb Paksé in northern Thailand, DNA analysis is recommended to be performed in all cases when peaks of Hb CS/Hb Paksé are detected on CE or HPLC.

Hb S [β6(A3)Glu→Val, GAG>GTG] is a β-globin gene variant that has a very low incidence in the Thai population. Coinheritance of Hb S and β(0)-thalassemia (β-thal) can result in severe clinical conditions. This study reports the case of a Thai patient with a compound heterozygosity for Hb S and β(0)-thal codon 17 (A>T). His hemoglobin (Hb), hematocrit (packed cell volume, PCV), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) levels were all less than the lower limits, while red cell distribution width (RDW) was higher than the upper limit. Levels of Hbs S, F and A(2) detected by high performance liquid chromatography (HPLC) were comparable to those from capillary electrophoresis (CE). As Hb S has a similar electrophoretic mobility and the HPLC profile is also similar to those of Hb Tak [β147, Term→Thr (+AC)] and Hb D-Punjab [β121(GH4)Glu→Gln, GAA>CAA], DNA sequencing was then performed. This was to detect β(0)-thal, and to differentiate Hb S from the Hb Tak and Hb D-Punjab mutations. The β(0)-thal codon 17 and Hb S mutations were detected indicating that coinheritance of these two mutations can be found in the Thai population. Therefore, to provide proper clinical management and genetic counseling of this rare case, DNA analysis should be performed in all cases when a peak at the S-window is detected by HPLC or CE.

The β-chain hemoglobin (Hb) variants interfere with the diagnosis of β-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a β-chain Hb variant frequently found in the Thai population, on β-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA(2) in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA(2) level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of β(0)-thalassemia codon 17, 41/42, and 71/72 mutations and β-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of β-thalassemia trait analyzed by CE but not by HPLC.

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is often missed by routine laboratory testing since its mRNA as well as gene product are unstable and presented at a low level in peripheral blood. This study aimed to analyze the efficacy of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) for detecting and quantifying Hb CS in 19 heterozygotes and 14 homozygotes with Hb CS as well as 10 Hb H-CS disease subjects who were detected by molecular analysis. In the CE electrophoregram, Hb CS was seen at zone 2 and was observed in all samples, while the chromatogram of Hb CS peaks was found in 26.32% heterozygotes, 42.86% homozygotes and 90% Hb H-CS disease subjects, respectively. In addition, the Hb CS levels in each group of subjects quantified by CE were significantly higher than those quantified by HPLC. Based on the CE method, the lowest Hb CS level was found in the heterozygotes, whereas the highest level was found in the Hb H-CS disease patients. Therefore, the CE method was superior to the HPLC method for detecting Hb CS. Furthermore, the level of Hb CS quantified by CE proved useful in screening heterozygotes and homozygotes with Hb CS as well as Hb H-CS disease.

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.

In order to provide a rapid method for identifying alpha-thalassemia 1 in a region with massive population and limited resources, we have tested a rapid screening strategy. Preliminary screening was done using a modified one tube osmotic fragility test (OF test) followed by RBC indices; Hb analysis and detection of alpha-thalassemia 1 with the Southeast Asian deletion (SEA type) were performed by PCR. One hundred and seventy-five adult Thai subjects were studied. Fifty-one of the 175 subjects (29.1%) were positive for a modified OF test. They all had significantly lower MCV and MCH but higher RDW-CV values as compared to the OF negative group. A successful identification of alpha-thalassemia 1 deletion using a direct PCR on cell lysates was demonstrated. Among the 51 OF-test-positive subjects, 7 were found to be alpha-thalassemia 1 carriers, 3 of whom were also carriers of Hb E. No alpha-thalassemia 1 was detected in the OF-test-negative group. A combination of a modified OF test and a direct PCR analysis on whole blood cell lysates would therefore provide an effective screening for alpha-thalassemia 1 in the regions where a program of prevention and control of the disease remains underserved., (Copyright 2002 S. Karger AG, Basel)