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.

Downregulation of miR-21 gene expression by CRE-Ter to modulate osteoclastogenesis: De Novo mechanism.

miR-21 expression stimulates osteoclast cells in the context of osteoclastogenesis. A previous report showed that NFκB-miR-21 pathway could serve as an innovative alternative to devise therapeutics for healing diabetic ulcers. Furthermore, our study demonstrated that a highly water-soluble curcuminoids-rich extract (CRE-Ter) inhibits osteoclastogenesis through NFκB pathway. The interplay between miR-21 and CRE-Ter in osteoclastogenesis has not yet been investigated. In this study, we examined the relation of CRE-Ter and miR-21 gene expression in receptor of the nuclear factor κB (NFκB) ligand (RANKL) - induced murine monocyte/macrophage RAW 264.7 cells, osteoclast cells, in osteoclastogenesis. Effect of CRE-Ter on generation of intracellular reactive oxygen species (ROS) was estimated by dichlorofluorescein diacetate (DCFH-DA). The results reveal that CRE-Ter reduced expression levels of miR-21 gene in osteoclasts. The inhibitory effects of CRE-Ter on in vitro osteoclastogenesis were evaluated by reduction in tartrate-resistant acid phosphatase (TRAP) content, and by reduction in expression levels of an osteoclast-specific gene, cathepsin K. Treatment of the osteoclast cells with CRE-Ter suppressed RANKL-induced NFκB activation including phospho-NFκB-p65, and phospho IκBα proteins. Western blot analysis revealed that NFκB inhibitor up-regulated CRE-Ter-promoted expression of phospho-NFκB-p65. In addition, CRE-Ter dose-dependently inhibited phospho-Akt expression. CRE-Ter also dose-dependently reduced DNA binding activity of NFκB and Akt as revealed by EMSA. ChIP assay revealed binding of NFκB-p65 to miR-21 promoters. In conclusion, our results demonstrate that CRE-Ter downregulates miR-21 gene expression in osteoclasts via a de novo mechanism, NFκB- Akt-miR-21 pathway.

High resolution melting analytical platform for rapid prenatal and postnatal diagnosis of ß-thalassemia common among Southeast Asian population.

BACKGROUND: High resolution melting (HRM) analysis is a powerful technology for scanning sequence alteration. We have applied this HRM assay to screen common ß-thalassemia mutations found among Southeast Asian population. METHODS: Known DNA samples with 8 common mutations were used in initial development of the methods including -28 A-G, codon 17 A-T, IVSI-1G-T, IVSI-5G-C, codon 26G-A (Hb E), codons 41/42 -TTCT, codons 71/72+A and IVSII-654 C-T. Further validation was done on 60 postnatal and 6 prenatal diagnoses of ß-thalassemia. RESULTS: Each mutation has specific HRM profile which could be used in rapid screening. Apart from those with DNA deletions, the results of HRM assay matched 100% with those of routine diagnosis made by routine allele specific PCR. In addition, the HRM assay could initially recognize three unknown mutations including a hitherto un-described one in Thai population. CONCLUSIONS: The established HRM assay should prove useful for rapid and high throughput platform for screening and prenatal diagnosis of ß-thalassemia common in the region.

A large cohort of ß(+)-thalassemia in Thailand: molecular, hematological and diagnostic considerations.

We report the molecular and hematological characteristics associated with a large cohort of ß(+)-thalassemia in Thailand. Study was done on 21,068 unrelated subjects referred to our center in northeast Thailand for hemoglobinopathies investigation. Among 21,068 subjects, 2637 (12.5%) were found to carry ß-thalassemia. Of these 2637 cases, 705 (26.7%) carried ß(+)-thalassemia with eight different mutations including 6 promoter mutations; NT-28 (A-G), NT-31 (A-G), NT-50 (G-A), NT-86 (C-G), NT-87 (C-A) and NT-90 (C-T) and two missense mutations; Hb Malay (codon 19; AAC-AGC) and Hb Dhonburi (codon 126; GTG-GGG). Hematological features of carriers with these ß(+)-thalassemia (n=528) were compared with those with ß(0)-thalassemia (n=309). Data for Hb E-ß(+)-thalassemia (n=177) were also presented along with Hb E-ß(0)-thalassemia in our series (n=94). All patients with Hb E-ß(+)-thalassemia were associated with mild thalassemia intermedia phenotypes. Most of the ß(+)-thalassemia carriers had elevated Hb A2 and mild hypochromic microcytosis, some demonstrated borderline MCV and MCH values which, could compromise carrier screening. Analysis of α/ß-globin mRNA ratio in representative cases with normal, Hb E trait, ß(+)-thalassemia trait, Hb Dhonburi trait and ß(0)-thalassemia trait demonstrated the average values of 1.1, 1.7, 2.1, 1.7 and 3.1, respectively which is helpful in identification and differentiation of the cases.

Molecular characterization of a ß-thalassemia intermedia patient presenting inferior vena cava thrombosis: interaction of the ß-globin erythroid Krüppel-like factor binding site mutation with Hb E and α(+)-thalassemia.

The molecular basis and hematological phenotype of adult Thai ß-thalassemia intermedia (ß-TI) patients encountered with inferior vena cava (IVC) thrombosis were investigated. Hematological and molecular analysis revealed a trait previously not described. The disease was caused by interaction of the ß(+)-thalassemia (ß(+)-thal) gene with the -90 (C > T) (HBB: c.-140C > T) transition within the erythroid Krüppel-like factor (EKLF) binding site of the ß-globin gene promoter with Hb E (HBB: c.79G > A) and α(+)-thalassemia (α(+)-thal). Hematological data of the patient were compared with those of heterozygous forms of these defects found in his family members and different genotype-phenotype interactions are illustrated. Globin gene haplotype analysis indicates an independent origin of this Thai ß(+)-thal gene. Accurate diagnoses as well as knowledge of genotype-phenotype relationships were required for providing appropriate management of such cases.

Prenatal and post-natal screening of ß-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography.

We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common ß-thalassemia mutations found in Thailand. The ß-globin gene was separately amplified by PCR on four different fragments covering eight most common ß-thalassemia mutations including nucleotide -28 A-G, codon 17 (A-T), IVSI-1 (G-T), IVSI-5 (G-C), codon 26 (G-A or Hb E), codons 41/42 (-TTCT), codons 71/72 (+A) and IVSII-654 (C-T). After PCR amplification, heteroduplex was generated by denaturation at 95 °C for 5 min followed by a slow reduction in temperature to 25 °C at 0.03 °C/s. Analysis of heteroduplex was done on an automated WAVE Nucleic Acid Fragment Analysis System. Specific DHPLC profile for each mutation was demonstrated which could be used in screening for all eight ß-thalassemia mutations. Further validation was done on 42 pre- and post-natal DNA samples which demonstrated 100 % accuracy as compared to the result obtained with conventional PCR assays. In a remaining case with an unknown mutation, a different DHPLC profile was noted on one of the amplified fragment. Further DNA sequencing of this fragment revealed a T-G transversion at the IVSI-116, a previously un-described mutation in Thai population. The DHPLC assay developed should prove useful for rapid screening of known and unknown ß-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.