Reliability of hemoglobin A2 value as measured by the Premier Resolution system for screening of ß-thalassemia carriers.

OBJECTIVES: Accurate quantification of hemoglobin (Hb) A2 is vital for diagnosing ß-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA2 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 HbA2 cutoff value for ß-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the ß- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing. RESULTS: HbA2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias, -0.33; r, 0.991), ß-thalassemia (bias, -0.27; r, 0.927), and non-ß-thalassemia carriers (bias, -0.36; r, 0.928). An HbA2 cutoff value of ≥4.0 % for ß-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 ß-Hb variant, Hb La Desirade [HBB:c.389C>T], associated with ß-thalassemia and co-inherited with a single α-globin gene. CONCLUSIONS: The Premier Resolution HPLC is a reliable and accurate method for routine ß-thalassemia carrier screening, aligning with existing CE methods.

Molecular understanding of unusual HbE-ß+-thalassemia with Hb phenotype similar to HbE heterozygote: simple and rapid differentiation using HbE levels.

BACKGROUND: Low HbF expression in HbE-ß+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the ß- 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-ß+-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 ß- and α-globin genes. DNA polymorphism at position -158 nt 5' to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-ß+-thalassemia from HbE heterozygotes were determined. RESULTS: Five ß+-thalassemia mutations trans to ßE-gene (ß-87(C>A), ß-31(A>G), ß-28(A>G), ß19(A>G), and ß126(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-ß+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-ß+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at -158 nt 5' to Gγ-globin was found to be negatively associated with HbF expression. CONCLUSIONS: HbE-ß+-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-ß+-thalassemia diagnosis.

HbE-ß⁺-thalassemia displays a wide range of HbF expression, which may lead to the misdiagnosis of HbE heterozygosity in patients whose Hb analysis shows HbE and HbA. α-Thalassemia may be a major factor associated with decreased secondary activation of HbF expression in the disease.HbE may be a potential indicator for effectively differentiating HbE-ß⁺-thalassemia from HbE heterozygotes.The high proportion and heterogeneity of α-thalassemia mutations found in patients with HbE-ß⁺-thalassemia evoke a complex thalassemia syndrome, requiring complete DNA analysis.

Hemoglobin profile and molecular characteristics of the complex interaction of hemoglobin Doi-Saket [α9(A7) asn > lys, HBA2:c.30C > a], a novel α2α1 hybrid globin variant, with hemoglobin E [ß26(B8) Glu > lys, HBB:c.79G > A] and deletional α+-thalassemia in a Thai family.

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 HbA2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α3.7 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 αDoi-Saket with ßE 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 -α3.7 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.

The Hb Doi-Saket is a novel α-globin variant mutation occurring in the α2-globin gene in cis to the -α^3.7 kb chromosome.The carrier of Hb Doi-Saket may present slight microcytosis and have severe clinical entities when it interacts with deletions in α-globin genes.Hb analysis with the HPLC system could completely separate Hb Doi-Saket and its derivative from other Hbs.

Effective screening of hemoglobin Constant Spring and hemoglobin Paksé with several forms of α- and ß-thalassemia in an area with a high prevalence and heterogeneity of thalassemia using capillary electrophoresis.

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 ß-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 A2 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.

Revisiting and updating molecular epidemiology of α-thalassemia mutations in Thailand using MLPA and new multiplex gap-PCR for nine α-thalassemia deletion.

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 α0-thalassemia (--CR or --SA deletion) with α+-thalassemia (-α3.7/-α4.2/αConstant Springα). 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 α0-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.

Phenotypic Expression of Known and Novel Hemoglobin A2-Variants, Hemoglobin A2-Mae Phrik [Delta 52(D3) Asp > Gly, HBD:c.158A > G], Associated with Hemoglobin E [Beta 26(B8) Glu > Lys, HBB:c.79G > A] in Thailand

The interactions of δ-globin variants with α- and ß-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 ß-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.

Development of a High Resolution Melting Curve Analysis for the Detection of Hemoglobin δ-Chain Variants in Thailand and Identification of Hb A2-Walsgrave [codon 52 (GAT>CAT), Asp→His; HBD:c.157G>C] in a Pregnant Woman from Southern Thailand.

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.

Hb Athens-Georgia (beta 40(C6) Arg > Lys, HBB:c.122G > A) with a single α-globin gene (Hb H disease) in a Thai family: molecular, hematological, and diagnostic aspects.

Interaction of structural hemoglobin (Hb) variants with α- or ß-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 ß-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 ß-globin gene was identical to the Hb A-Ga (HBB:c.122G > A). Interestingly, α-thal-1 (SEA deletion) and α-thal-2 (-α3.7 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 (ßA-Ga/ßA, -SEA/-α3.7) had moderate anemia and was markedly hypochromic with microcytic red blood cells (RBCs). The father (ßA-Ga/ßA, -SEA/αα) presented mild microcytic anemia, while normal hematology was observed in the I-2 who was heterozygous for Hb Athens-Georgia (ßA-Ga/ßA, 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 ß-haplotype [+ - - - - - +]. These findings were in accordance with the previous conclusion that this variant is a non-pathological ß-Hb variant.

Molecular Characterization and Hematological Aspects of Hb E-Myanmar [ß26(B8)Glu→Lys and ß65(E9)Lys→Asn, HBB: c.[79G>A;198G>C]): A Novel ß-Thalassemic Hemoglobin Variant.

Hb E [ß26(B8)Glu→Lys, GAG>AAG, HBB: c.79G>A] is an inherited thalassemic ß-globin variant that favors the Hb E-ß-thalassemia (ß-thal) syndrome when interacting with the ß-thal gene. However, hemoglobin (Hb) variants carrying Hb E in combination with another variant on the same ß gene are rare. We recently studied a 29-year-old pregnant woman, initially diagnosed as a ß-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 A2 but no Hb E or abnormal Hb peaks, with a markedly elevated Hb A2 level (6.4%) reaching the accepted range (4.0-10.0%) for ß-thal trait. DNA analysis identified a GAG>AAG transition at codon 26 of the ß-globin gene that is responsible for Hb E, and an AAG>AAC mutation at codon 65 in cis on the ß-globin chain resulting in a lysine to asparagine substitution. These two mutations led to the formation of a novel variant, namely Hb E-Myanmar, ß26(B8)Glu→Lys and ß65(E9)Lys→Asn, HBB: c.[79G>A;198G>C]. Moreover, a heterozygous α-thalassemia-2 (α-thal-2) [-α3.7 (rightward)] deletion was also observed. Hb E-Myanmar is a doubly substituted ß-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 ß-globin chain.

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.

Molecular Characterization of ß- 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 ß-thalassemia (ß-thal) carriers. However, some ß-thal carriers have been misdiagnosed as their Hb A2 levels are below 4.0%. In addition, coinheritance of α-thalassemia (α-thal) and ß-thal might affect Hb A2 levels. Therefore, the aim of this study was to investigate the mutations of ß- 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 ß-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 ß-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 (ß4)-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 ß-thal carriers coinheriting α-thal were observed, and five of them carried Hb H disease. High prevalence of both α- and ß-thal in subjects with borderline Hb A2 levels suggested that molecular diagnosis of α- and ß-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, ß-thal carriers who coinherited Hb H disease might have reduced Hb A2 levels, leading to a misdiagnosis of ß-thal in analysis programs.

α-Thalassemia Intermedia Results from Interactions of Unstable Hb Prato [α31(B12)Arg→Ser (HBA1 or HBA2 c.96G>T or C)] with the α-Thalassemia-1 [- -SEA (Southeast Asian)] Deletion in Thailand.

The clinical consequences of many abnormal hemoglobins (Hbs) interacting with α- or ß-thalassemia (α- or ß-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.

Molecular spectrum of Hb H disease and characterization of rare deletional α-thalassemia found in Thailand.

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/-α3.7; n = 312), (-SEA/-α4.2; n = 26), (-THAI/-α3.7; n = 1) and two non-deletional types (-SEA/αCSα; n = 131), (-SEA/αPakséα; n = 2). Herein, we firstly report a rare genotype of Hb H disease with (-SA/-α3.7; n = 1) that has not been documented in Thailand, and rare genotypes related to (-SEA/-α16.6; n = 1), and (-SEA/αQSα; 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.

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

In this study, Hb A2 variants and their association with α- and ß-thalassemia (α- and ß-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). ß0-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 ß-thal, two (8.7%) carried Hb A2' and one (4.3%) carried Hb A2-Lampang. All three samples doubly heterozygous for Hb A2-Melbourne/ß0-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 ß-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 ß-thal. ß-Globin haplotype analysis showed an association with a single ß-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.

Analysis of Deletional Hb H Diseases in Samples with Hb A2-Hb H and Hb A2-Hb Bart's on Capillary Electrophoresis.

The capillary electrophoresis (CE) system allows the quantification of Hb Bart's (γ4) and Hb H (ß4) 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 α0-thalassemia (α0-thal) [- -SEA (Southeast Asian) and - -THAI (Thailand)] deletions and the α+-thal [-α3.7 (rightward) and -α4.2 (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 A2-Hb H phenotype on CE electrophoregrams presented the genotype of - -SEA/-α3.7, while the - -SEA/-α4.2 made up the remainder. The - -SEA/-α3.7 genotype was also found in all six samples (100.0%) with Hb A2-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.

Coinheritance of Hb A2-Melbourne (HBD: c.130G>A) and Hb E (HBB: c.79G>A) in Laos and Simultaneous High Resolution Melt Detection of Hb A2-Melbourne and Hb A2-Lampang (HBD: c.142G>A) in a Single Tube.

We report the molecular and hematological identifications of a Hb A2 variant [coinheritance of Hb A2-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 A2, 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 (GAG>AAG) causing a glutamic acid to lysine substitution corresponding to Hb A2-Melbourne. The origin of Hb A2-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 A2-Melbourne and Hb A2-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 A2 variant in the routine molecular setting.

A Case Report of Compound Heterozygosity for ß0/ß+-Thalassemia Resulting from under Diagnosed ß-Thalassemia Found in a Hb A'2 Sample.

Hb A'2 (or Hb B2) (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 ß0 codon 17 (A>T; HBB: c.52A>T) and ß+ IVS II-654 (C>T; HBB: c.316-197C>T). Under diagnosed ß-thalassemia (ß-thal) in her father, who carries Hb A'2 and a heterozygous ß0 codon 17 mutation, and the mother, who carries a heterozygous ß+ 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 ß-thal in Hb A'2 samples. This case highlights the importance of Hb A'2 in prenatal diagnosis (PND). Thus, molecular analysis for ß-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 A2 was equal or higher than 4.0%.

Diagnosis of Compound Heterozygous Hb Tak/ß-Thalassemia and HbD-Punjab/ß-Thalassemia by HbA2 Levels on Capillary Electrophoresis.

A misdiagnosis of ß-thalassemia carrier in samples with Hb Tak and HbD-Punjab, the ß-variants, can be a cause of inappropriate genetic counseling thus having a new case of ß-thalassemia major. A capillary electrophoresis (CE) is very efficient in separating and quantifying HbA2. In this study, HbA2 levels of samples which were doubted for compound heterozygous Hb Tak/ß-thalassemia or heterozygous HbD-Punjab/ß-thalassemia were measured and compared between CE and high performance liquid chromatography (HPLC). The molecular confirmation for Hb Tak, HbD-Punjab and ß-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/ß-thalassemia and one for HbD-Punjab/ß-thalassemia. The elevated HbA2 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 HbA2 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 ß-thalassemia.