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.
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References
Weatherall DJ, Clegg JB (2001) Inherited haemoglobin disorders: an increasing global health problem. Bull World Health Organ 79:704–712
Rund D, Rachmilewitz E (2005) Beta-thalassemia. N Engl J Med 353:1135–1146
Fucharoen S, Winichagoon P (1992) Thalassemia in Southeast Asia: problem and strategy for prevention and control. Southeast Asian J Trop Med Public Health 23:647–655
Nuntakarn L, Fucharoen S, Fucharoen G, Sanchaisuriya K, Jetsrisuparb A, Wiangnon S (2009) Molecular, hematological and clinical aspects of thalassemia major and thalassemia intermedia associated with Hb E-beta-thalassemia in northeast Thailand. Blood Cells Mol Dis 42:32–35
Yamsri S, Sanchaisuriya K, Fucharoen G, Sae-Ung N, Ratanasiri T, Fucharoen S (2010) Prevention of severe thalassemia in northeast Thailand: 16 years of experience at a single university center. Prenat Diagn 30:540–546
Yamsri S, Sanchaisuriya K, Fucharoen G, Sae-Ung N, Fucharoen S (2011) Genotype and phenotype characterization in a large cohort of β-thalassemia heterozygote with different forms of α-thalassemia in northeast Thailand. Blood Cells Mol Dis 47:120–124
Losekoot M, Fodde R, Harteveld CL, van Heeren H, Giordano PC, Bernini LF (1990) Denaturing gradient gel electrophoresis and direct sequencing of PCR amplified genomic DNA: a rapid and reliable diagnostic approach to beta thalassaemia. Br J Haematol 76:269–274
Fortina P, Dotti G, Conant R et al (1992) Detection of the most common mutations causing beta-thalassemia in Mediterraneans using a multiplex amplification refractory mutation system (MARMS). PCR Methods Appl 2:163–166
Sutcharitchan P, Saiki R, Fucharoen S, Winichagoon P, Erlich H, Embury SH (1995) Reverse dot-blot detection of Thai beta-thalassaemia mutations. Br J Haematol 90:809–816
Xiao W, Oefner PJ (2001) Denaturing high-performance liquid chromatography: a review. Hum Mutat 17:439–474
Fucharoen S, Fucharoen G, Ratanasiri T, Jetsrisuparb A, Fukumaki Y (1994) A simple non radioactive assay for hemoglobin E gene in prenatal diagnosis. Clin Chim Acta 229:197–203
Fucharoen S, Fucharoen G, Ratanasiri T, Jetsrisuparb A, Fukumaki Y (1995) A simple non-radioactive method for detecting β-thalassemia/Hb E disease: application to prenatal diagnosis. Southeast Asian J Trop Med Public Health 26(suppl 1):278–281
Siriratmanawong N, Fucharoen G, Sanchaisuriya K, Ratanasiri T, Fucharoen S (2001) Rapid and simultaneous detection of β-thalassemia and α-thalassemia 1 (SEA type) in prenatal diagnosis of complex thalassemia syndrome. Clin Biochem 34:377–380
Fucharoen G, Fucharoen S, Sanchaisuriya K et al (2002) Frequency distribution and haplotypic heterogeneity of βE-globin gene among eight minority groups of northeast Thailand. Hum Hered 53:18–22
Srivorakun H, Fucharoen G, Sae-Ung N, Sanchaisuriya K, Ratanasiri T, Fucharoen S (2009) Analysis of fetal blood using capillary electrophoresis system: a simple method for prenatal diagnosis of severe thalassemia diseases. Eur J Haematol 83:57–65
Vrettou C, Traeger-Synodinos J, Tzetis M, Malamis G, Kanavakis E (2003) Rapid screening of multiple beta-globin gene mutations by real-time PCR on the Light Cycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes. Clin Chem 49:769–776
Herrmann MG, Dobrowolski SF, Wittwer CT (2000) Rapid beta-globin genotyping by multiplexing probe melting temperature and color. Clin Chem 46:425–428
Bournazos SN, Tserga A, Patrinos GP, Papadakis MN (2007) A versatile denaturing HPLC approach for human beta-globin gene mutation screening. Am J Hematol 82:168–170
Colosimo A, Guida V, De Luca A et al (2002) Reliability of DHPLC in mutational screening of beta-globin (HBB) alleles. Hum Mutat 19:287–295
Li Q, Li LY, Huang SW et al (2008) Rapid genotyping of known mutations and polymorphisms in beta-globin gene based on the DHPLC profile patterns of homoduplexes and heteroduplexes. Clin Biochem 41:681–687
Fucharoen S, Winichagoon P, Wisedpanichkit R et al (1998) Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC. Clin Chem 44:740–748
Metherall JE, Collins FS, Pan J, Weissman SM, Forget BG (1986) Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron. EMBO J 5:2551–2557
Flint J, Harding RM, Boyce AJ, Clegg JB (1993) The population genetics of the haemoglobinopathies. Baillere’s Clin Haematol 6:215–262
Timbs AT, Rugless MJ, Gallienne AE, Haywood AM, Henderson SJ, Old JM (2012) Prenatal diagnosis of hemoglobinopathies by pyrosequencing: a more sensitive and rapid approach to fetal genotyping. Hemoglobin 36:144–150
Harteveld CL, Kleanthous M, Traeger-Synodinos J (2009) Prenatal diagnosis of hemoglobin disorders: present and future strategies. Clin Biochem 42:1767–1779
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This work was supported by a grant from the National Research University (NRU) program of Khon Kaen University. TP is supported by the Royal Golden Jubilee PhD program (PHD/0137/2551) of the Thailand Research Fund (TRF), Thailand.
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Prajantasen, T., Fucharoen, S., Fucharoen, G. et al. Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography. Mol Biol Rep 40, 3173–3179 (2013). https://doi.org/10.1007/s11033-012-2391-4
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DOI: https://doi.org/10.1007/s11033-012-2391-4