Retrospective study and implementation of a low-cost LAMP-turbidimetric assay for screening α0-thalassemia (SEA deletion): preventing and controlling Hb Bart's hydrops fetalis syndrome in Thailand.

Homozygous α0-thalassemia (SEA deletion) or Hb Bart's hydrops fetalis syndrome is a significant public health issue in Thailand and Southeast Asia. A prevention and control program has been implemented in this region. This study focuses on retrospective laboratory data collected between January 2021 and April 2023 at a single center. Additionally, we developed a low-cost LAMP-turbidimetric assay to propose in the screening strategy. A total of 3,623 samples underwent screening tests (MCV, MCH, and DCIP), including 1,658 couple screenings (84.25%) and 310 single pregnant screenings (15.75%). Negative screenings, which did not require further investigation, were found in 75.51% for couple screenings and 46.58% for single pregnant screenings. At hemoglobin (Hb) analysis identified 129 couples which had fetuses at risk of severe thalassemia, whereas molecular analysis during the retrospective period revealed 210 samples with different genotypes. These remaining samples were validated using the low-cost LAMP-turbidimetric assay to detect α0-thalassemia (SEA deletion). The developed LAMP turbidimetric assay demonstrated a sensitivity and specificity of 100% (36/36 × 100) and 97.7% (170/174 × 100), respectively, when compared with gap-PCR. Furthermore, we propose a strategy involving the addition of the low-cost LAMP-turbidimetric assay before performing the gold standard. This strategy represents a cost-saving of USD 2,608 based on 210 samples that required DNA analysis. Finally, the developed LAMP turbidimetric assays offer advantages such as reduced time, workload, cost savings, no need for highly developed instruments, and a straightforward interpreting process. Therefore, implementation of LAMP assays into routine settings would be improve the efficiency of prevention and control program for severe thalassemia disease in this region.

Direct cord blood LAMP colorimetric phenol red assay for detecting α0-thalassemia (SEA deletion); the validation and post-natal screening in Thailand.

Post-natal or newborn screening for thalassemia and hemoglobinopathies is useful for genetic counseling and managing thalassemia in children. We characterized thalassemia genotypes in newborns from the eastern part of Thailand. The results demonstrated a high heterogeneity of thalassemia and hemoglobinopathies with seventeen genotypes. We focused on α0- thalassemia (Southeast Asian [SEA] deletion) in this study. We developed and validated the loop-mediated isothermal amplification (LAMP) colorimetric assay for detecting α0- thalassemia (SEA deletion) using simple direct cord blood sampling compared to genomic DNA. A total of 160 cord blood samples were evaluated with the LAMP assay. The sensitivity and specificity of the LAMP colorimetric assay for α0-thalassemia (SEA deletion) using direct cord blood showed 100% (6/6 x 100) and 98.05% (151/154 x 100) whereas, genomic DNA showed 100% (6/6 x 100) and 100% (154/154 x 100), respectively. Moreover, we demonstrated other simple screening tools for α0-thalassemia with %Hb Bart's, MCV, and MCH values and found that these parameters were not diagnostic in our samples. The direct cord blood with colorimetric LAMP assay is simple, rapid, and does not require a post-LAMP step compared to conventional PCR. These techniques could be applied in post-natal or large population screening for α0-thalassemia (SEA deletion). Finally, this could support early prevention of complications, early management, genetic counseling for α-thalassemia disease in children, or a long-term prevention and control program of severe thalassemia in Thailand.

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.

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.

Genetic Background Studies of Eight Common Beta Thalassemia Mutations in Thailand Using ß-Globin Gene Haplotype and Phylogenetic Analysis.

Single nucleotide polymorphisms are informative for haplotype analysis associated with genetic background and clinical linkage studies of ß-thalassemia mutations. Hence, the aim of this study was to investigate five polymorphisms (codon 2 (C/T), IVS II-16 (C/G), IVS II-74 (G/T), IVS II-81 (C/T) and the Hinf I (T/A) polymorphism) on the ß-globin gene, related to eight common ß-thalassemia mutations in Thailand, including NT-28 (A > G), codon 17 (A > T), codon 19 (A > G), HbE (G > A), IVS I-1 (G > C), IVS I-5 (G > C), codon 41/42 (-TTCT) and IVS II-654 (C > T). The strongest LD (100%) between the ß-thalassemia mutation allele and all five SNPs was found in NT-28 (A > G), codon 17 (A > T) and codon 19 (A > G). In the haplotype analysis, we found three haplotypes (H1, H2 and H7) related to Hb E, whereas we only found two haplotypes related to codon 41/42 (-TTCT) (H1, H3) and IVS I-1 (G > C) (H3, H4). Of interest is the finding relating to a single haplotype in the remaining ß-thalassemia mutations. Furthermore, phylogenetic tree analysis revealed three clusters of these common ß-thalassemia mutations in the Thai population and enabled us to determine the origin of these mutations. Here, we present the results of our study, including four intragenic polymorphisms and the finding that the Hinf I polymorphism could be informative in genetic background analysis, population studies and for predicting the severity of ß-thalassemia in Thailand.

Loop-mediated isothermal amplification (LAMP) colorimetric phenol red assay for rapid identification of α0-thalassemia: Application to population screening and prenatal diagnosis.

BACKGROUND: Identification of α0-thalassemia (SEA and THAI deletions) is essential in preventing and controlling of severe thalassemia diseases. We have developed the LAMP colorimetric assays for the detection of these two thalassemia defects and validated them in population screening and prenatal diagnosis. METHODS: Three LAMP colorimetric assays specific for α0-thalassemia (SEA deletion), α0-thalassemia (THAI deletion) and normal DNA sequence were developed. These assays were validated on 341 subjects who had initial thalassemia screening positive and various thalassemia genotypes. Prenatal diagnosis of α0-thalassemia (SEA deletion) was done on 33 fetuses at risk of having Hb Bart's hydrops fetalis syndrome. RESULTS: The LAMP colorimetric assays for α0-thalassemia (SEA and THAI deletions) could be clearly interpreted by naked eyes. The assay for α0-thalassemia (SEA deletion) showed a 100% (62/62 x 100) sensitivity and 98.2% (274/279 x 100) specificity whereas, that of the α0-thalassemia (THAI deletion) showed 100% (1/1 x 100) sensitivity and 99.7% (339/340 x 100) specificity. We obtained a 100% concordant prenatal diagnosis results using LAMP assays of α0-thalassemia (SEA deletion) in 33 fetuses as compared to the conventional PCR analysis. CONCLUSIONS: The LAMP colorimetric assays developed are simple, rapid, and do not require sophisticated equipment. Inclusion of the LAMP tests in the existing screening protocol significantly reduce the screening cost and the molecular analysis workload, which should prove useful in the prevention and control program of hemoglobinopathies in the region.

Rapid molecular diagnostics of large deletional ß0-thalassemia (3.5 kb and 45 kb) using colorimetric LAMP in various thalassemia genotypes.

BACKGROUND: ß-thalassemia is an inherited disorder that is reported worldwide. Two common ß0-thalassemia mutations (3.5 kb and 45 kb deletions) are prevalent in Southeast Asia and Thailand. Identification of these defects is essential to population screening and prenatal diagnosis. We aimed to develop colorimetric LAMP based on a phenol red indicator and validate it on various thalassemia genotypes. METHOD: Colorimetric LAMP assays for detecting ß0-thalassemia 3.5- and 45-kb deletions were developed and validated on 254 routine clinical samples. The results of the assays could be interpreted by the naked eye and compared with the gold standard gap-PCR. RESULTS: A total of 254 samples related to seven phenotypes and 27 different genotype groups showed 100% concordance between the colorimetric LAMP assays and gap-PCR for detecting ß0-thalassemia (3.5- and 45-kb deletions). The sensitivity, specificity, NPV, and PPV were calculated as 100% for both ß0-thalassemia 3.5- and 45-kb deletion detection. The comparison of the usefulness of colorimetric LAMP assays and conventional methods was demonstrated in this study. CONCLUSIONS: The developed colorimetric LAMP assays are rapid, simple, and highly cost effective and can be interpreted by the naked eye. These assays should be applied for screening deletional ß0-thalassemia in routine settings or small community hospitals in remote areas where thalassemia is highly heterogeneous.

Molecular Spectrum of ß-Thalassemia Mutations in Central to Eastern Thailand.

The aim of this study was to determine the molecular spectrum of ß-thalassemia (ß-thal) mutations in eastern Thailand. We identified ß-thal mutations using allele specific-polymerase chain reaction (ASPCR) and direct DNA sequencing. We found 18 different ß-thal mutations in a total of 191 unrelated subjects. Six common ß-thal mutations comprised 86.91% of all the mutations, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) (35.60%), codon 17 (A>T) (HBB: c.52A>T) (18.85%), -28 (A>G) (HBB: c.-78A>G) (15.71%), IVS-II-654 (C>T) (HBB: c.316-197C>T) (6.28%), IVS-I-1 (G>T) (HBB: c.92+1G>T) (5.76%) and codon 19 (A>G) (HBB:(c.59A>G) (4.71%). In addition, a novel 60 kb deletion in two unrelated cases was characterized and initially suspected to originate from eastern Thailand. Moreover, we demonstrated the molecular spectrum of recent ß-thal mutations in Thailand, and data from this study were compared with five reference laboratory centers in Thailand. This study is the first to identify the comprehensive molecular spectrum of ß-thal mutations in eastern Thailand, information that may be essential for screening, genetic counseling and prenatal diagnosis (PND) in this region.

Genetics background of ß-thalassemia (3.5 kb deletion) in Southern Thailand: Haplotype analysis using novel reverse dot blot hybridization.

ß-thalassemia (ß-thal) (3.5 kb deletion or NC_000011.10:g.5224302-5227791del3490bp) is a common mutation in southern Thailand. This study aimed to determine genetic diversity in subjects with ß-thal (3.5 kb deletion) alleles and to ascertain the origin of this mutation using haplotype and phylogenetic analysis. The study was carried out on members of the southern Thai population, including 45 normal individuals, 116 heterozygous ß-thal (3.5 kb deletion) and one homozygous ß-thal (3.5 kb deletion). The 5'-haplotype in ß-globin gene cluster was examined using newly developed reverse dot blot hybridization (RDB) and compared with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results showed 100% concordance between the haplotype patterns of these two methods. From a total of 324 chromosomes, nine haplotypes were segregated. Haplotype H2 (+ - - - -) was the predominant haplotype observed in all 118 ß-thal (3.5 kb deletion) chromosomes, which revealed a single origin. The phylogenetic tree demonstrated that ß-thal (3.5 kb deletion) has an older genetic defect in this region. Moreover, the developed RDB is simple, less time-consuming, inexpensive, and does not restriction enzyme digestion.

Rapid Molecular Detection for Differentiation of Homozygous HbE and ß0-Thalassemia/HbE in Samples Related With HbE >80% and Variable HbF Levels.

OBJECTIVE: To validate a novel rapid molecular testing method for differentiation of homozygous hemoglobin (Hb)E and HbE/ß 0-thalassemia genotypes using multiplex melt curve combined with high-resolution melt (HRM) analysis in a single test tube. METHODS: All 10 genotypes contained (ß N/ß N; n = 95), (ß N/ß 3.5-kb; n = 71), (ß N/ß 45-kb; n = 28), (ß N/ß E; n = 10), (ß E/ß 3.5-kb; n = 6), (ß E/ß 45-kb; n = 4), (ß E/ß 41/42; n = 28), (ß E/ß 17; n = 9), (ß E/ß IVSI#1; n = 6), and (ß E/ß E; n = 76) were recruited for validation. A proposed strategy for rapid differentiation of ß 0-thalassemia/HbE disease and homozygous Hb E in specimens with HbE greater than 80% and variable HbF levels was demonstrated. RESULTS: In the validation method, all genotypes showed 100% concordance, compared with the conventional reverse dot blot (RDB) and gap-polymerase chain reaction (PCR) methods. CONCLUSIONS: Our newly developed method could be useful in routine laboratory settings. The method is rapid, simple, and cost effective; does not require a post-PCR step; and can be applied in routine settings.

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.

A novel SNP rs11759328 on Rho GTPase-activating protein 18 gene is associated with the expression of Hb F in hemoglobin E-related disorders.

Hemoglobin (Hb) F has a modulatory effect on the clinical phenotype of ß-thalassemia disease. High expression of Hb F in Hb E-related disorders has been noted, but the mechanism is not well understood. We have examined the association of a novel SNP rs11759328 on ARHGAP 18 gene and other known modulators with a variability of Hb F in Hb E-related disorders. Genotyping of SNP rs11759328 (G/A) was performed based on high-resolution melting analysis. The rs11759328 (A allele) was shown to be significantly associated with Hb F levels (p < 0.05) in heterozygous and homozygous Hb E. High levels of Hb F in both heterozygous and homozygous Hb E were also found to be associated with SNPs in the study of other modifying genes including KLF 1 mutation, rs7482144 (Gγ-XmnI), rs4895441, rs9399137 of (HBS1L-MYB), and rs4671393 (BCL11A). Multivariate analysis showed that KLF1 mutation and SNP rs11759328 (GA) (ARHGAP18) modulated Hb F expression in heterozygous Hb E. For homozygous Hb E, this was found to be related to five modifying factors, i.e., KLF1 mutation, rs4895441 (GG), rs9399137 (CC), rs4671393 (AA), and rs4671393 (GA). These results indicate that a novel SNP rs11759328 is a genetically modifying factor associated with increased Hb F in Hb E disorder.

Strong Linkage of the Single Nucleotide Polymorphism rs77308790 with an α0-Thalassemia (- -SEA deletion) Allele and Application for Double-Check Diagnosis of Hb Bart's Hydrops Fetalis Syndrome in Thailand.

The α0-thalassemia (α0-thal) [- -SEA (Southeast Asian) deletion] is highly prevalent in Southeast Asia and South China. The linkage between the single nucleotide polymorphism (SNP) rs77308790 and the - -SEA deletion was reported in the Chinese population. This study reported the genotype of SNP rs77308790 using the high resolution melting (HRM) curve analysis in the Thai population and the application for double-checking diagnosis of Hb Bart's (γ4) hydrops fetalis syndrome. A total of 202 samples, including α0-thal carriers (- -SEA/αα) (n = 99) and wild-type (n = 103), was recruited. Minor allele frequency (MAF) of SNP rs77308790 (T allele) represented a significant difference (p<0.001) between carrier (- -SEA deletion) (MAF 0.455) and wild-type (MAF 0.039). The T allele of SNP rs77308790 showed a strong linkage with the - -SEA deletion allele [correlation coefficient between pairs of loci (D' = 1)] based on constructed random samples (CRSs) in Thais. Moreover, worldwide populations, based on the 1000Genomes database, also found the T allele to be less than 1.0%. For providing a double-checked diagnosis, two SNP (rs3760053, rs77308790) genotypes showed 100.0% concordance with a conventional gap-polymerase chain reaction (gap-PCR) method in nine families at-risk for Hb Bart's hydrops fetalis. The double-checked diagnosis based on the two SNPs (rs3760053, rs77308790) is suitable for implementation in routine diagnosis of Hb Bart's hydrops fetalis syndrome. Furthermore, our HRM analysis system can be amplified with a small amount of fetal DNA and could avoid allele dropouts.

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.

Molecular analysis of haemoglobin E in Southeast Asian populations.

Haemoglobin (Hb) E is the most common Hb variant in Asia where its gene frequency approaches 0.3 in some areas. We studied genetic background of Hb E genes among Southeast Asian populations. This study examined ß-globin gene haplotypes linked to haemoglobin E (Hb E) in diverse groups of Southeast Asian populations. The study was conducted on southern Thai (22 alleles), Cambodian (84 alleles), Laotian (120 alleles), Vietnamese (87 alleles) and Burmese (one allele) subjects. Results were compared with those of previous studies in northeast Thailand, the Yunnan of China, West India and Europe. Ten different haplotypes were observed. The four most common haplotypes were haplotypes 1 (- + - + + + -) and 2 (+ - - - - + -) on chromosomes with framework 2 and haplotypes 6 (- + - + + - +) and 7 (+ - - - - - +) on chromosomes with framework 3 variety. Phylogenetic analysis indicated that haplotype 1 is a relatively recent haplotype found in all populations, whereas haplotype 6 is found predominately in Cambodians. The results indicate that at least two genetic origins of Hb E are responsible for the high prevalence and spread of Hb E among Southeast Asian populations.

Genetic origin of α0-thalassemia (SEA deletion) in Southeast Asian populations and application to accurate prenatal diagnosis of Hb Bart's hydrops fetalis syndrome.

α0-thalassemia of SEA deletion (-SEA) is common among Southeast Asian and Chinese. Using haplotype and phylogenetic analyses, we examined the origin of this defect in Southeast Asian populations. Study was done on both normal and α0-thalassemia alleles in 3 ethnic groups including 96 Thai, 52 Laotian and 21 Cambodian. Five SNPs encompassing the (-SEA) including (rs3760053 T>G), (rs1211375 A>C), (rs3918352 A>G), (rs1203974 A>G) and (rs11248914 C>T) were examined using high-resolution melting assays. It was found that 94.0% of Thai, 100% of Laotian and 100% of Cambodian α0-thalassemia alleles were linked to the same haplotype: the haplotype H4 (AAGC), representing an Asian specific origin. An G allele of the (rs3760053) was found to be in strong linkage disequilibrium with the α0-thalassemia allele in these populations. A multiplex PCR assay was developed to detect simultaneously the (-SEA) allele and genotyping of a linked (rs3760053) to improve accuracy of prenatal diagnosis of α0-thalassemia. Application of this multiplex PCR assay for routine prenatal diagnosis of α0-thalassemia in 12 families revealed a 100% concordant result with conventional gap-PCR assay. Therefore, a single genetic origin is responsible for the spread and high prevalence of the (-SEA) in the region. The multiplex PCR assay developed should provide a double-check PCR system for more accurate diagnosis and allow the monitoring of possible maternal contamination at prenatal diagnosis of this important genetic disorder.

Screening of (-SEA) α-thalassaemia using an immunochromatographic strip assay for the ζ-globin chain in a population with a high prevalence and heterogeneity of haemoglobinopathies.

AIMS: The presence of the ζ-globin chain is a good marker of (--SEA) α0-thalassaemia. We evaluated an immunochromatographic (IC) strip assay for ζ-globin in screening for (--SEA) α0-thalassaemia in a population with a high prevalence and heterogeneity of haemoglobinopathies. METHODS: The study was carried out on 300 screen positive blood samples of Thai individuals. The IC strip assay for the ζ-globin chain was performed on all samples. The results were interpreted with thalassaemia genotyping using standard haemoglobin and DNA analyses. RESULTS: Several thalassaemia genotypes were noted. Among the 300 subjects investigated, 79 had a positive IC strip assay for ζ-globin and (--SEA) α0-thalassaemia was identified in 40 of them. No (--SEA) α0-thalassaemia was detected in the remaining 39 samples with a positive IC strip test result or in the 221 samples with a negative IC strip test result. Further DNA analysis identified α+-thalassaemia in 25 of the 39 (--SEA) α0-thalassaemia negative samples. Using this IC strip assay in combination with a conventional screening protocol for (--SEA) α0-thalassaemia could provide sensitivity and specificity of 100% and 90.4%, respectively. CONCLUSIONS: IC strip assay for ζ-globin is simple, rapid and does not require sophisticated equipment. Use of this test in addition to the existing screening protocol could detect potential (--SEA) α0-thalassaemia leading to a significant reduction in the workload of DNA analysis. This could be used in areas where haemoglobinopathies are prevalent and heterogeneous but molecular testing is not available.

Hemoglobin Constant Spring among Southeast Asian Populations: Haplotypic Heterogeneities and Phylogenetic Analysis.

BACKGROUND: Hemoglobin Constant Spring (Hb CS) is an abnormal Hb caused by a mutation at the termination codon of α2-globin gene found commonly among Southeast Asian and Chinese people. Association of Hb CS with α°-thalassemia leads to a thalassemia intermedia syndrome commonly encountered in the region. We report chromosome background and addressed genetic origins of Hb CS observed in a large cohort of Hb CS among Southeast Asian populations. MATERIALS AND METHODS: A study was done on 102 Vietnamese (aged 15-49 year-old) and 40 Laotian (aged 18-39 year-old) subjects with Hb CS and results compared with 120 Hb CS genes in Thailand. Hematological parameters were recorded and Hb analysis was performed using capillary electrophoresis. Hb CS mutation and thalassemia genotypes were defined by DNA analysis. Six DNA polymorphisms within α-globin gene cluster including 5'Xba I, Bgl I, Inter-zeta HVR, AccI, RsaI and αPstI 3', were determined using PCR-RFLP assay. RESULTS: Nine different genotypes of Hb CS were observed. In contrast to the Thai Hb CS alleles which are mostly linked to haplotype (+-S + + -), most of the Vietnamese and the Laotian Hb CS genes were associated with haplotype (+-M + + -), both of which are different from that of the European Hb CS. CONCLUSIONS: Hb CS is commonly found in combination with other thalassemias among Southeast Asian populations. Accurate genotyping of the cases requires both hematologic and DNA analyses. At least two independent origins are associated with the Hb CS gene which could indirectly explain the high prevalence of this Hb variant in the region.