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.

Anemia, iron deficiency, and thalassemia among the Thai population inhabiting at the Thailand-Lao PDR-Cambodia triangle.

Anemia is a major public health problem in many areas of Southeast Asia. Ascertaining anemia and defining its underlying causes is essential for providing appropriate care, management, and establishment of a control program. Limited studies on these have been carried out on people living at the borders of Thailand, Lao PDR, and Cambodia. This cross-sectional study was done in four areas along the borders of Thailand, Lao PDR, and Cambodia. Blood specimens were collected from subjects aged 15-18 years in four districts including Kantharalak, Si Sa Ket province (n = 36), Nam Khun (n = 109), Nam Yuen (n = 98), and Na Chaluai (n = 128), Ubon Ratchathani province, Thailand. RBC parameters were recorded, and serum ferritin (SF) level was measured. Diagnosis of thalassemia and hemoglobinopathies was based on hemoglobin (Hb) and DNA analyses. Measurement of C-reactive protein was performed to exclude false-negative result of iron deficiency. The prevalence of anemia was found to be 25.1%. ID accounted for only 10.5%. Various types of thalassemia were identified in 67.7% of the subjects. The overall prevalence of thalassemia included 3.5% α0-thalassemia, 0.8% ß-thalassemia, 47.7% Hb E, and 53.6% α+-thalassemia. The proportions of ID, thalassemia and combined ID and thalassemia among anemic subjects were 6.5%, 66.6%, and 20.4%, respectively. The results indicate that thalassemia and hemoglobinopathies rather than ID are major causes of anemia in Thailand-Lao PDR-Cambodia triangle. This information should prove useful for implementing an anemia control program in the regions.

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.

Five Variable Number of Tandem Repeats Loci (D17S5, APOB, TPO Intron 10, IL-1α Intron 6, and CIAS1) in Thais and Application in the Prenatal Diagnostic Laboratory.

Background: Prenatal diagnosis of genetic disease requires DNA analysis of fetal tissue of a responsible gene. Accurate diagnosis is useful for the appropriate management of pregnancy. However, maternal contamination of fetal specimens poses a high preanalytical risk of prenatal misdiagnosis. We have examined five variable number of tandem repeat (VNTR) polymorphisms for use in monitoring potential maternal contamination. Materials and Methods: A study was conducted to examine the heterozygosities of five VNTR loci including, D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 in 200 unrelated Thai subjects and applied to the monitoring of maternal contamination in 22 families at risk of having fetuses with severe thalassemia. Results: The heterozygosities of D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 VNTRs were 59.5, 19.5, 66.0, 35.5, and 42.0%, respectively. Therefore, the TPO intron 10 and D17S5 loci were chosen for prenatal diagnosis of thalassemia in 22 families. Analyses of these VNTRs demonstrated an increase of informative data from 59.1% provided by the routine D1S80 VNTR analysis to 90.9%. Conclusions: The VNTR diagnostic procedure described above is simple, cost-effective, rapid, and does not require the use of sophisticated instruments; it should prove useful in the prenatal diagnosis of thalassemia.

Micromapping of Thalassemia and Hemoglobinopathies Among Laos, Khmer, Suay and Yer Ethnic Groups Residing in Lower Northeastern Thailand.

Northeastern (NE) Thailand is one of the areas with a prevalence of thalassemias and hemoglobinopathies. Data on the prevalence of the diseases in minorities in the region has been limited. This study aimed to survey the thalassemias and hemoglobinopathies that take into account ethnicity. Four ethnic groups, including Laos (n = 162), Khmer (n = 145), Suay (n = 134), and Yer (n = 101) inhabiting the lower region of NE Thailand, were selected to represent the study populations. The results demonstrated that an extremely high prevalence of Hb E (HBB: c.79G>A) (>50.0%) was observed in the Khmer, Suay and Yer ethnic groups. The highest prevalence of α+-thalassemia (α+-thal) [-α3.7 (rightward)] deletion was found in the Khmer ethnic group (48.28%). The -α4.2 (leftward) deletion (α+-thal) was restricted to the Yer ethnic group. Yer and Suay had a high incidence of Hb Constant Spring (Hb CS; HBA2: c.427T>C) as well as Hb Paksé (HBA2: c.429A>T). As the prevalence of α0-thalassemia (α0-thal) is relatively high in Suay (7.46%), couples who are members of Suay ethnic population should be urged to undergo hematological screening before planning a pregnancy to control the Hb Bart's hydrops fetalis. Micromapping of thalassemias and hemoglobinopathies herein described will be helpful in genetic counseling and public education campaigns, which should be carried out in appropriate languages, with exhibitions at the village levels. This information will be of benefit for the long-term effort to reduce the burden of severe thalassemia disease in the region.

Dominant ß-thalassaemia with unusually high Hb A2 and Hb F caused by ßCD121(-G) (HBB:c.364delG) in exon 3 of ß-globin gene.

We describe a dominant ß-thalassaemia caused by a deletion of G at nucleotide position 364 in exon 3 of the ß-globin gene. The heterozygosity of this mutation was found in a 36-year-old Thai patient who had moderate hypochromic microcytic anaemia with haemolytic blood picture. Haemoglobin (Hb) analysis revealed relatively higher Hbs A2 (6.8%) and F (4.7%) as compared with those of ß0-thalassaemia (n=278) and ß+-thalassaemia (n=55) carriers in our series. Secondary structure prediction of the elongated ß-globin chain showed that the α-helix at the C-terminal is disrupted dramatically by the random coil and ß-sheet, which should result in a highly unstable ß-globin variant, undetectable in peripheral blood and a dominant clinical phenotypic feature.

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.

Molecular characteristics of α+-thalassemia (3.7 kb deletion) in Southeast Asia: Molecular subtypes, haplotypic heterogeneity, multiple founder effects and laboratory diagnostics.

OBJECTIVE: The 3.7 kb deletion (-α3.7) is the most common form of α+-thalassemia found in multiple populations which can be classified into three subtypes. In order not to mis-identify it, the molecular information within each population is required. We have addressed this in northeast Thai and Laos populations. METHODS: Screening for α+-thalassemia was initially done on 1192 adult Thai subjects. In addition, 77 chromosomes of Thai newborns and 26 chromosomes of Laos with -α3.7 α+-thalassemia were also examined. All subjects were screened for -α3.7 α+-thalassemia and subtyped by PCR-RFLP assay. Exact deletion breakpoint of each -α3.7 subtype was determined by DNA sequencing. α-Globin gene haplotypes were determined. RESULTS: The proportions of -α3.7 subtypes found in 216 Thai -α3.7 chromosomes were 94.9% for -α3.7I, 4.2% for α3.7II and 0.9% for -α3.7III. All 26 Laos -α3.7 chromosomes were of -α3.7I variety. At least six α-globin gene haplotypes were associated with the -α3.7I α+-thalassemia. CONCLUSION: All -α3.7 subtypes were observed among Southeast Asian population. Haplotype analysis indicated a multiple origin of this common disorder in the region. A multiplex PCR assay has been developed for simultaneous detection of all subtypes of -α3.7 α+-thalassemia as well as other α+-thalassemia found in the region including -α4.2 α+-thalassemia, Hb Constant Spring and Hb Paksé.

Fetal blood sampling in prenatal diagnosis of thalassemia at late pregnancy.

Fetal blood sampling is a procedure that involves the drawing of a blood sample from the umbilical vein of the umbilical cord, which can be performed after 18 weeks gestation. Fetal blood sampling is a preferable method for prenatal diagnosis of thalassemia in second trimester or late pregnancy. Additionally, it is suggested to be performed in cases in which mosaicisms are identified by amniocentesis or chorionic villus sampling (CVS), areas where DNA analysis is not available, and when mutations of the parents are not known. Laboratory steps regarding prenatal diagnosis by fetal blood sampling were summarized, including the ensuring of fetal origin, determination of red blood cell parameters, fetal hemoglobin analysis, and finally fetal DNA analysis. The objective of this review is to present an overview of procedures in terms of benefits, laboratory interpretations, and some limitations.

Genetic heterogeneity of hemoglobin AEBart's disease: a large cohort data from a single referral center in northeast Thailand.

AEBart's disease is a thalassemia intermedia usually characterized by the interaction of α(0)-thalassemia with either deletional or non-deletional α(+)-thalassemia in Hb E heterozygote. Genotypic and phenotypic features are heterogeneous. We studied the hematologic and molecular characteristics of this disease in a cohort of 173 Thai patients encountered at our center in northeast Thailand. Hemoglobin and DNA analyses identified patients with deletional AEBart's disease (n=84), Hb Constant Spring AEBart's disease (n=81), Hb Paksé-AEBart's disease (n=5), AEBart's disease with codon 30 mutation (n=1) and two hitherto un-described forms of AEBart's disease due to interaction of Hb E heterozygote and α(0)-thalassemia with the -α(16.6)kb deletional α(+)-thalassemia (n=1) and Hb Q-Thailand (n=1). Different phenotypic expression of these AEBart's diseases with low Hb, Hct and MCV and increased RDW values with marked reduction in Hb E levels were observed. It was found that all these forms of AEBart's disease showed similar thalassemia intermedia phenotypes but those with non-deletional forms were relatively more anemic. Our data confirm that in such area with high prevalence of hemoglobinopathies such as Southeast Asia, identification of rare thalassemia alleles in a thalassemia intermedia patient should not be ignored. Careful consideration of different phenotypic expression may help in providing presumptive diagnosis of this disease where access to molecular testing is limited. However, molecular diagnostic is useful for predicting the clinical outcome and improving genetic counseling of these complex hemoglobinopathies.

Fetal red blood cell parameters in thalassemia and hemoglobinopathies.

INTRODUCTION: With the lack of fetal blood specimens in routine practice, little is known about red blood cell (RBC) parameters of fetuses with various thalassemia syndromes. This study aimed to describe these in various forms of thalassemia. MATERIALS AND METHODS: The study was performed on 93 fetal blood specimens obtained from pregnant women by cordocentesis during 18-24 weeks of gestation. RBC parameters were recorded on automated analyzer. Hemoglobin (Hb) and DNA analyses were performed for definite genotyping. RESULTS: No significant difference in RBC parameters was observed between non-thalassemic fetuses and those with ß-thalassemia trait, Hb E trait, homozygous Hb E and ß-thalassemia/Hb E disease. However, in those with α(0)-thalassemia trait and double heterozygous α(0)-thalassemia/Hb E, slight reduction in mean corpuscular volume (MCV) was noted. Fetuses with the Hb H disease showed significant reductions in Hb, MCV and mean corpuscular Hb (MCH). Marked reductions in Hb, hematocrit, MCH and mean cell Hb concentration and increased RBC distribution width with numerous nucleated RBC were clearly observed in Hb Bart's hydrops fetalis. CONCLUSION: Simple analysis of fetal RBC parameters is useful for making presumptive prenatal diagnosis of α-thalassemia syndromes including Hb H disease and Hb Bart's hydrops fetalis which can then be confirmed by Hb and DNA analyses.

A proficiency testing program of hemoglobin analysis in prevention and control of severe hemoglobinopathies in Thailand.

BACKGROUND: No external quality assessment program for hemoglobin (Hb) analysis in the prevention and control of thalassemia has been established in Thailand. To improve the first line provisional diagnostics, the first proficiency testing (PT) program has been established. METHODS: External Hb controls prepared at our center were sent to Hb analysis laboratories all over the country. Three cycles per year were performed in 2010 and 2011. In each cycle, two control samples with corresponding hematological parameters, designated as husband and his pregnant wife were supplied for Hb analysis. Each member analyzed the control samples in their routine practices. The results of Hb analysis, laboratory interpretation and risk assessment of the expected fetus for severe thalassemia diseases targeted for prevention and control were entered into the report form and sent back to our center. Participants reports were analyzed and classified into four different quality groups; Excellent (when all the three parameters are correct), Good (correct Hb analysis and interpretation but incorrect risk assessment), Fair (correct Hb analysis but incorrect interpretation and risk assessment) and Needs improvement (incorrect Hb analysis). RESULTS: It was found that most participants could report correct Hb types and quantifications but some misinterpretations and risk assessments were noted. These were clearly seen when control samples with more complexity were supplied. CONCLUSIONS: These results indicate a further improvement is required in the laboratory interpretation and knowledge of the laboratory diagnosis of thalassemia. The established system should facilitate the prevention and control program of thalassemia in the region.

Evaluation of the URIT-2900 automated hematology analyzer for screening of thalassemia and hemoglobinopathies in Southeast Asian populations.

BACKGROUND: The effectiveness of the URIT-2900 Hematology Analyzer for screening of hemoglobinopathies commonly found in Southeast Asian populations was examined. METHODS: Appropriate cut-off values of MCV and MCH for screening of α(0) and ß thalassemias were derived from the receiver operator characteristic curve conducted initially on 279 subjects with various thalassemia genotypes. Validation was performed additionally in a cohort of another unrelated 313 subjects. RESULTS: The best cut off values of MCV and MCH were found to be 78fL and 27pg, respectively. Using these cut off values in combination with the dichlorophenolindophenol test in screening of α(0) thalassemia, ß thalassemia and Hb E in a cohort study revealed 100% sensitivity, 79.6% specificity, 80.0% positive predictive value and 100% negative predictive value. CONCLUSION: The combined blood cell counting using the URIT-2900 Automated Hematology Analyzer and dichlorophenolindophenol test is suitable for population screening of thalassemia and hemoglobinopathies in Southeast Asia.

Hemoglobin Q-Thailand related disorders: origin, molecular, hematological and diagnostic aspects.

We describe the molecular and hematological profiles of thalassemia syndromes caused by interactions of hemoglobin (Hb) Q-Thailand [α74(EF3) Asp-His] and various hemoglobinopathies found in 52 unrelated adult Thai subjects. Ten genotypes including several previously undescribed conditions were observed, which were classified into 4 groups. Group I included 26 Hb Q-Thailand heterozygotes and a homozygotous subject. Group II included subjects with Hb Q-Thailand and other α-thalassemia alleles in trans including 1 compound Hb Q-Thailand/α(+)-thalassemia (-α(3.7)), 2 Hb Q-Thailand/Hb Constant Spring disease and 6 Hb H/Q-Thailand disease. The average levels of Hb Q-Thailand were found to be 29.8%, 82.3%, 34.7%, 49.2-49.3% and 79.4%, respectively. Both Hbs Bart's and H were observed in addition to Hb Q-Thailand in all 6 cases with Hb Q-H disease but not in a homozygous Hb Q-Thailand. Group III included 7 double heterozygotes for Hb Q-Thailand/Hb E, 3 Hb Q-Thailand/Hb E/α(+)-thalassemia (-α(3.7)), 3 heterozygous Hb Q-Thailand/homozygous Hb E and 1 triple heterozygote for Hb Q-Thailand/Hb Constant Spring/Hb E. In this group, Hbs E (α(A)(2)ß(E)(2)), Q-Thailand (α(QT)(2)ß(A)(2)) and QE (α(QT)(2)ß(E)(2)) were observed on both HPLC and capillary electrophoresis. The Hb QE, rather than Hb Q-Thailand, was detected in all 3 cases with heterozygous Hb Q-Thailand and homozygous Hb E. The remaining two cases in group 4 were double heterozygotes for Hb Q-Thailand and ß(0)-thalassemia in which Hb Q-Thailand, elevated Hb A(2) (α(A)(2)δ(2)), and Hb QA(2) (α(QT)(2)δ(2)) were detected. DNA analysis identified the Hb Q-Thailand mutation (α74: GAC-CAC) and the linked (-α(4.2)) in all cases. Analysis of α-globin gene haplotype provided the first evidence of a single origin of this Hb variant in Thai population.

Accurate prenatal diagnosis of Hb Bart's hydrops fetalis in daily practice with a double-check PCR system.

Hemoglobin (Hb) Bart's hydrops fetalis is a fatal condition associated with homozygous alpha(0)-thalassemia. Prenatal diagnosis of the disease is usually done by gap-PCR; however, misdiagnosis can occur with allelic dropout. Diagnosis using more than one method is preferred. We describe a double-check PCR assay for accurate prenatal diagnosis. The study was conducted on 64 fetuses at risk of homozygous alpha(0)-thalassemia encountered at our routine thalassemia diagnosis laboratory. Chorionic villus sample (CVS), amniotic fluid or fetal blood specimens were obtained from pregnant women at risk and analyzed by two PCR methods. In the first method, the SEA alpha(0)-thalassemia deletion of parents and fetuses were determined by gap-PCR routinely run in our laboratory. In another method, two specific fragments located 5' to the zeta(2) gene (XbaI fragment) and the alpha(2)-globin gene (RsaI fragment) together with the gap-PCR fragment were multiply co-amplified to determine the presence or absence of normal and alpha(0)-thalassemia alleles. The molecular diagnosis of alpha(0)-thalassemia was possible in all 64 fetuses using the two PCR approaches. The final diagnoses included 13 normal, 29 unaffected heterozygote and 22 homozygote alpha(0)-thalassemia fetuses.The two PCR assays disclosed no discordant result in the diagnosis of the Hb Bart's hydrops fetalis caused by alpha(0)-thalassemia.The combined PCR assay for gap-PCR, zeta(2) XbaI and alpha(2) RsaI fragments, described here, is simple, accurate and applicable in the prenatal diagnosis of Hb Bart's hydrops fetalis in a routine setting.