International Journal of Hematology

, Volume 106, Issue 6, pp 757–764 | Cite as

High-resolution melting analysis for prenatal diagnosis of beta-thalassemia in northern Thailand

  • Pimlak Charoenkwan
  • Supatra Sirichotiyakul
  • Arunee Phusua
  • Sudjai Suanta
  • Kanda Fanhchaksai
  • Rattika Sae-Tung
  • Torpong Sanguansermsri
Original Article

Abstract

High-resolution melting (HRM) analysis is a rapid mutation analysis which assesses the pattern of reduction of fluorescence signal after subjecting the amplified PCR product with saturated fluorescence dye to an increasing temperature. We used HRM analysis for prenatal diagnosis of beta-thalassemia disease in northern Thailand. Five PCR–HRM protocols were used to detect point mutations in five different segments of the beta-globin gene, and one protocol to detect the 3.4 kb beta-globin deletion. We sought to characterize the mutations in carriers and to enable prenatal diagnosis in 126 couples at risk of having a fetus with beta-thalassemia disease. The protocols identified 18 common mutations causing beta-thalassemia, including the rare codon 132 (A–T) mutation. Each mutation showed a specific HRM pattern and all results were in concordance with those from direct DNA sequencing or gap-PCR methods. In cases of beta-thalassemia disease resulting from homozygosity for a mutation or compound heterozygosity for two mutations on the same amplified segment, the HRM patterns were different to those of a single mutation and were specific for each combination. HRM analysis is a simple and useful method for mutation identification in beta-thalassemia carriers and prenatal diagnosis of beta-thalassemia in northern Thailand.

Keywords

Beta-thalassemia High-resolution melting HRM analysis Molecular diagnosis Prenatal diagnosis 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Higgs DR, Thein SL, Wood WG. Thalassaemia: classification, genetics and relationship to other inherited disorders of haemoglobin. In: Weatherall DJ, Clegg JB, editors. The thalassaemia syndromes. Oxford: Blackwell Science; 2001. p. 121–32.Google Scholar
  2. 2.
    Cao A, Galanello R. Beta-thalassemia. Genet Med. 2010;12:61–76.CrossRefPubMedGoogle Scholar
  3. 3.
    Fucharoen S, Winichagoon P. Thalassemia in Southeast Asia: problems and strategy for prevention and control. Southeast Asian J Trop Med Public Health. 1992;23:647–55.PubMedGoogle Scholar
  4. 4.
    Fucharoen S, Weatherall DJ. The hemoglobin E thalassemias. Cold Spring Harb Perspect Med. 2012;2 (pii: a011734).Google Scholar
  5. 5.
    Tongsong T, Charoenkwan P, Sirivatanapa P, Wanapirak C, Piyamongkol W, Sirichotiyakul S, et al. Effectiveness of the model for prenatal control of severe thalassemia. Prenat Diagn. 2013;33:477–83.CrossRefPubMedGoogle Scholar
  6. 6.
    Lin M, Jiao JW, Zhan XH, Zhan XF, Pan MC, Wang JL, et al. High resolution melting analysis: a rapid screening and typing tool for common beta-thalassemia mutation in Chinese population. PLoS One. 2014;9:e102243.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pornprasert S, Phusua A, Suanta S, Saetung R, Sanguansermsri T. Detection of alpha-thalassemia-1 Southeast Asian type using real-time gap-PCR with SYBR Green1 and high resolution melting analysis. Eur J Haematol. 2008;80:510–4.CrossRefPubMedGoogle Scholar
  8. 8.
    Prajantasen T, Fucharoen S, Fucharoen G. High resolution melting analytical platform for rapid prenatal and postnatal diagnosis of beta-thalassemia common among Southeast Asian population. Clin Chim Acta. 2015;441:56–62.CrossRefPubMedGoogle Scholar
  9. 9.
    Prathomtanapong P, Pornprasert S, Phusua A, Suanta S, Saetung R, Sanguansermsri T. Detection and identification of beta-thalassemia 3.5 kb deletion by SYBR Green1 and high resolution melting analysis. Eur J Haematol. 2009;82:159–60.CrossRefPubMedGoogle Scholar
  10. 10.
    Saetung R, Ongchai S, Charoenkwan P, Sanguansermsri T. Genotyping of beta thalassemia trait by high-resolution DNA melting analysis. Southeast Asian J Trop Med Public Health. 2013;44:1055–64.PubMedGoogle Scholar
  11. 11.
    Shih HC, Er TK, Chang TJ, Chang YS, Liu TC, Chang JG. Rapid identification of HBB gene mutations by high-resolution melting analysis. Clin Biochem. 2009;42:1667–76.CrossRefPubMedGoogle Scholar
  12. 12.
    Sirichotiyakul S, Saetung R, Sanguansermsri T. Analysis of beta-thalassemia mutations in northern Thailand using an automated fluorescence DNA sequencing technique. Hemoglobin. 2003;27:89–95.CrossRefPubMedGoogle Scholar
  13. 13.
    Wasi P, Pootrakul S, Pootrakul P, Pravatmuang P, Winichagoon P, Fucharoen S. Thalassemia in Thailand. Ann N Y Acad Sci. 1980;344:352–63.CrossRefPubMedGoogle Scholar
  14. 14.
    Fukumaki Y, Fucharoen S, Fucharoen G, Okamoto N, Ichinose M, Jetsrisuparb A, et al. Molecular heterogeneity of beta-thalassemia in Thailand. Southeast Asian J Trop Med Public Health. 1992;23(Suppl 2):14–21.PubMedGoogle Scholar
  15. 15.
    Thein SL, Winichagoon P, Hesketh C, Best S, Fucharoen S, Wasi P, et al. The molecular basis of beta-thalassemia in Thailand: application to prenatal diagnosis. Am J Hum Genet. 1990;47:369–75.PubMedPubMedCentralGoogle Scholar
  16. 16.
    Winichagoon P, Fucharoen S, Wilairat P, Chihara K, Fukumaki Y, Wasi P. Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease. Biochim Biophys Acta. 1992;1139:280–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Fucharoen S, Fucharoen G, Sriroongrueng W, Laosombat V, Jetsrisuparb A, Prasatkaew S, et al. Molecular basis of beta-thalassemia in Thailand: analysis of beta-thalassemia mutations using the polymerase chain reaction. Hum Genet. 1989;84:41–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Laosombat V, Fucharoen SP, Panich V, Fucharoen G, Wongchanchailert M, Sriroongrueng W, et al. Molecular basis of beta thalassemia in the south of Thailand. Am J Hematol. 1992;41:194–8.CrossRefPubMedGoogle Scholar
  19. 19.
    Boonyawat B, Monsereenusorn C, Traivaree C. Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study. Appl Clin Genet. 2014;7:253–8.PubMedPubMedCentralGoogle Scholar
  20. 20.
    Nopparatana C, Saechan V, Nopparatana C, Pornpatkul M, Panich V, Fukumaki Y. A novel 105 basepair deletion causing beta(0)-thalassemia in members of a Thai family. Am J Hematol. 1999;61:1–4.CrossRefPubMedGoogle Scholar
  21. 21.
    Sanguansermsri T, Pape M, Laig M, Hundrieser J, Flatz G. Beta zero-thalassemia in a Thai family is caused by a 3.4 kb deletion including the entire beta-globin gene. Hemoglobin. 1990;14:157–68.CrossRefPubMedGoogle Scholar
  22. 22.
    Charoenkwan P, Teerachaimahit P, Sanguansermsri T. The correlation of alpha-globin gene mutations and the XmnI polymorphism with clinical severity of Hb E/beta-thalassemia. Hemoglobin. 2014;38:335–8.CrossRefPubMedGoogle Scholar
  23. 23.
    Mirasena S, Shimbhu D, Sanguansermsri M, Sanguansermsri T. Detection of beta-thalassemia mutations using a multiplex amplification refractory mutation system assay. Hemoglobin. 2008;32:403–9.CrossRefPubMedGoogle Scholar
  24. 24.
    Old JM, Khan SN, Verma I, Fucharoen S, Kleanthous M, Ioannou P, et al. A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction. Hemoglobin. 2001;25:397–407.CrossRefPubMedGoogle Scholar
  25. 25.
    Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem. 2004;50:1748–54.CrossRefPubMedGoogle Scholar
  26. 26.
    Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003;49:853–60.CrossRefPubMedGoogle Scholar
  27. 27.
    Gudnason H, Dufva M, Bang DD, Wolff A. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucl Acids Res. 2007;35:e127.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Frischknecht H, Troxler H, Greiner J, Hengartner H, Dutly F. Compound heterozygosity for Hb S [beta6(A3)GluVal, GAG–>GTG] and a new thalassemic mutation [beta132(H10)Lys–>term, AAA–>TAA] detected in a family from West Africa. Hemoglobin. 2008;32:309–13.CrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2017

Authors and Affiliations

  1. 1.Department of Pediatrics, Faculty of MedicineChiang Mai UniversityChiang MaiThailand
  2. 2.Department of Obstetrics and Gynecology, Faculty of MedicineChiang Mai UniversityChiang MaiThailand
  3. 3.Thalassemia Research Unit, Institute of Human GeneticsUniversity of PhayaoPhayaoThailand

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