Background: Mutations in the FLT3 gene are associated with acute myeloid leukemia (AML). FLT3 mutations have been iden-tified in approximately 30% of de novo AML patients, particularly those with typical karyotype and inferior prog-nosis. Therefore, we examined the frequencies of an internal tandem duplication (ITD) and missense mutations of the FLT3 gene and their associated clinical characteristics in patients with AML in northeast Thailand. Material/Methods: The leftover bone marrow and/or peripheral blood specimens of 65 newly diagnosed AML patients recruited from Srinagarind Hospital, Khon Kaen University, northeast Thailand, between January 2020 and May 2021 were included in this study. FLT3-ITD and FLT3-tyrosine kinase domain (TKD) were amplified using PCR-related techniques. Results: The frequencies of FLT3-ITD and TKD were found to be 16.9% (11/65) and 10.8% (7/65), respectively. One patient had ITD and TKD mutations. The white blood cell count and peripheral blast percentage of FLT3-ITD-positive patients were statistically significantly higher than those of the FLT3-wild type patients, while the pe-ripheral blast percentage of FLT3-TKD-positive patients was significantly lower. No other clinical characteristics among FLT3-positive and FLT3-wild-type patients were observed. DNA sequencing identified 4 FLT3-TKD mu-tations. The c.2504A>T; Asp835Val and c.2503G>C; Asp835His mutations were predicted as pathogenic mu-tations while the 2 novel mutations, c.2508C>A; Ile836= and c.2508C>G; Ile836Met were predicted as neutral mutations. Conclusions: This study showed for the first time that FLT3-TKD mutation is common among northeast Thai AML patients. The data should prove useful for selecting efficacious targeted treatment plans for the patients.

Objective: Prenatal and postnatal diagnosis of hemoglobin E-β 0-thalassemia can be made using polymerase chain reaction (PCR) analysis mostly on purified DNA. We have establihed a direct amplification method without DNA extraction on whole blood (WB) and amniotic fluid (AF) specimens to diagnose the disease.Methods: Three reactions of WB PCR assays and 7 reactions of AF PCR tests were developed for postnatal and prenatal diagnosis, respectively. Assays were validated against routine tests in a blinded trial.Results: The results showed 100% concordance with routine DNA PCR assays. Among 309 β-thalassemia carriers, 191 patients (61.8%) carried common β-thalassemia mutations. Among 448 AF specimens, 116 (25.9%) fetuses were found to be affected, 247 (55.1%) fetuses were carriers, and 85 (19%) fetuses were unaffected.Conclusion: We found that WB and AF PCR assays are simple, rapid, and reliable. The developed techniques could be applicable in routine settings.

Hemoglobin Constant Spring (Hb CS) and Hb Pakse' (PS) are the common non-deletional α⁺-thalassemia found in Thailand. These two variants can cause severe thalassemia syndromes, especially in fetus and neonate. Molecular diagnosis is the only confirmatory method because Hb CS and Hb PS are usually missed by routine screening and Hb analysis. Therefore, we aimed to develop rapid direct PCR for the diagnosis of Hb CS and PS genes. Multiplex direct PCR assays for identifying the Hb CS and PS genes in whole blood (WB) and amniotic fluid (AF) specimens were developed. The assays were firstly validated on 290 unrelated whole blood specimens. Hb CS and PS carriers were identified in 67 (23.1%) and 6 (2.1%) cases, respectively. A 100% concordant result as compared to routine PCR assay was observed. The direct PCR assays have been applied successfully for prenatal diagnosis in two families. The result showed that the fetuses were affected by homozygous Hb CS and compound heterozygous Hb CS/Hb PS. Accurate prenatal diagnosis of these families was observed using the newly developed assays. These assays should be applicable in routine thalassemia diagnostics as well as in the large-scale screening of Hb CS and PS in the region.

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.

Hemoglobin Bart's hydrops fetalis is the most severe form of α-thalassemia, caused by homozygous α⁰-thalassemia, which is highly prevalent in Southeast Asian countries. Simple and rapid identification of α⁰-thalassemia carrier and prenatal diagnosis of Hemoglobin Bart's hydrops is essential in the region. We have developed a multiplex RPA assay for simple detection of α⁰-thalassemia (SEA and THAI deletions) and tested it in carrier detection (n = 125) and prenatal diagnosis of Hb Bart's hydrops fetalis syndrome (n = 30). The sensitivity, specificity, positive predictive value, and negative predictive value for detecting α⁰-thalassemia carriers were 100% for both SEA and THAI deletions. The assay correctly identified 42 carriers of α⁰-thalassemia (SEA deletion), 4 carriers of α⁰-thalassemia (THAI deletion), and 79 non-carriers. For prenatal diagnosis, the results of RPA revealed a 100% concordance (30/30) with the conventional gap-PCR analysis. The multiplex RPA assay is a rapid, reliable, and cost-effective method for diagnosing α⁰-thalassemia-related disorders in routine clinical settings. This assay has the potential to significantly contribute to the prevention and control of Hb Bart's hydrops fetalis in the region.