title: Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography.
abstract: We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common β-thalassemia mutations found in Thailand. The β-globin gene was separately amplified by PCR on four different fragments covering eight most common β-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 β-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 β-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.
authors.affiliation: Biomedical Science Program, Graduate School, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Thanet
authors.initials: T
authors.lastname: Prajantasen
authors.forename: Supan
authors.initials: S
authors.lastname: Fucharoen
authors.forename: Goonnapa
authors.initials: G
authors.lastname: Fucharoen
authors.forename: Nirut
authors.initials: N
authors.lastname: Siriratmanawong
authors.forename: Charnchai
authors.initials: C
authors.lastname: Pinmuang-Ngam
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005260:Female; D006446:Hemoglobin E / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009154:Mutation; D011247:Pregnancy; D011296:Prenatal Diagnosis; D013785:Thailand; D055544:beta-Globins / Q000235:genetics* / Q000302:isolation & purification; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D055544:beta-Globins; D006446:Hemoglobin E