PCR-DHPLC for Non-Invasive Prenatal Evaluation of the IVS-II-1 (G>A) Mutation in Fetal DNA of β-Thalassemia Major: A Pilot Diagnostic Performance Study
-
Mandana Zafari
,
Taher Sadeghi
,
Sara Banihashemi
,
Amirreza Hashemi
,
Maryam Bakhtiyari
,
Mehrnoush Kowsaryan
,
Pooria Gill
,
Hadis Musavi
Abstract
β-thalassemia is one of the most common autosomal recessive single-gene disorders, particularly in high-prevalence regions. This study aimed to explore the feasibility and diagnostic performance of polymerase chain reaction-denaturing high-performance liquid chromatography (PCR-DHPLC) for non-invasive prenatal detection of the IVS-II-1 (G>A) mutation in fetal DNA. Fifty pregnant women at risk of carrying a fetus with β-thalassemia major were enrolled. Cell-free DNA was extracted from maternal plasma and analyzed using high-resolution melting (HRM) real-time PCR, followed by PCR amplification. Amplicons were assessed by electrophoresis, purified using a PCR purification kit, and subsequently analyzed by DHPLC under predefined buffer and temperature conditions to detect the IVS-II-1 (G>A) mutation. DHPLC analysis identified 13 true positive, 11 true negative, 19 false negative, and 7 false positive cases compared with the invasive reference method. The calculated sensitivity and specificity were 40.6% and 61.1%, respectively, indicating limited diagnostic performance in the current setting. PCR-DHPLC does not provide sufficient sensitivity and specificity for reliable non-invasive prenatal detection of the IVS-II-1 (G>A) mutation in β-thalassemia major. Further methodological optimization and extended mutation panels in larger cohorts are necessary to assess its potential for clinical validation.
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10. Almomani AA, Ala’a SS, Atoom AM, Abdel MBA, Alhmoud JF. Evaluation of the validity of the pre-marriage mean corpuscular volume value as a predictive test for b-thalassemia carrier status. J Med Biochem 2023;42:195-201.
11. Grosso M, Sessa R, Puzone S, Storino MR, Izzo P. Molecular basis of thalassemia. Anemia 2012;2012:341580.
12. Rahimi Z, Muniz A, Parsian A. Detection of responsible mutations for beta thalassemia in the Kermanshah Province of Iran using PCR-based techniques. Mol Biol Rep 2010;37:149-54.
13. Jaafari Z, Sadidi N, Abdolahinia Z, Shahesmaeili A. Prevalence of depression among Iranian patients with beta-thalassemia major: a systematic review and meta-analysis. Iran J Med Sci 2022;47:15-24.
14. El-Fadaly N, Abd-Elhameed A, Abd-Elbar E, El-Shanshory M. Accuracy of reverse dot-blot PCR in detection of different β-globin gene mutations. Indian J Hematol Blood Transfus 2016;32:239-43.
15. Mahdieh N, Rabbani B. Beta thalassemia in 31,734 cases with HBB gene mutations: pathogenic and structural analysis of the common mutations; Iran as the crossroads of the Middle East. Blood Rev 2016;30:493-508.
16. Banan M, Bayat H, Azarkeivan A, Mohammadparast S, Kamali K, Farashi S, et al. The Xmn I and BCL11A single nucleotide polymorphisms may help predict hydroxyurea response in Iranian β-thalassemia patients. Hemoglobin 2012;36:371-80.
17. Epstein CJ, Golbus MS. Prenatal diagnosis of genetic diseases. Am Sci 1977;65:703-11.
18. Wilson RD, Gagnon A, Audibert F, Campagnolo C, Carroll J, Brock JA, et al. Prenatal diagnosis procedures and techniques to obtain a diagnostic fetal specimen or tissue: maternal and fetal risks and benefits. J Obstet Gynaecol Can 2015;37:656-68.
19. Sherlock JK. Minimally invasive prenatal diagnosis of inherited disorders employing trophoblastic cells shed into the endocervical canal [dissertation]. London: University College London; 1999.
20. Monni G, Zoppi MA, Iuculano A, Piras A, Arras M. Invasive or non-invasive prenatal genetic diagnosis? J Perinat Med 2014;42:545-8.
21. Shekhawat DS, Sharma C, Singh K, Singh P, Bhardwaj A, Patwa P. Critical appraisal of droplet digital polymerase chain reaction application for noninvasive prenatal testing. Congenit Anom (Kyoto) 2022;62:188-97.
22. Hanson B, Paternoster B, Povarnitsyn N, Scotchman E, Chitty L, Chandler N. Non-invasive prenatal diagnosis (NIPD): current and emerging technologies. Extracell Vesicles Circ Nucleic Acids 2023;4:3-17.
23. Zafari M, Gill P, Kowsaryan M, Alipour A, Banihashemi A. High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers. J Matern Fetal Neonatal Med 2016;29:3323-8.
24. Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: A review. Hum Mutat 2001;17:439-74.
25. Prajantasen T, Fucharoen S, Fucharoen G, Siriratmanawong N, Pinmuang-Ngam C. Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography. Mol Biol Rep 2013;40:3173-9.
26. Yenilmez ED, Tuli A, Evrüke İC. Noninvasive prenatal diagnosis experience in the Çukurova Region of Southern Turkey: detecting paternal mutations of sickle cell anemia and β-thalassemia in cell-free fetal DNA using high-resolution melting analysis. Prenat Diagn 2013;33:1054-62.
27. Tungwiwat W, Fucharoen G, Fucharoen S, Ratanasiri T, Sanchaisuriya K, Sae-Ung N. Application of maternal plasma DNA analysis for noninvasive prenatal diagnosis of Hb E-β-thalassemia. Transl Res 2007;150:319-25.
28. Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield. PLoS One 2011;6:e25202.
29. Raymond FL, Whittaker J, Jenkins L, Lench N, Chitty LS. Molecular prenatal diagnosis: the impact of modern technologies. Prenat Diagn 2010;30:674-81.
30. Chiu RW, Chan KA, Gao Y, Lau VY, Zheng W, Leung TY, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A 2008;105:20458-63.
31. Treff NR, Tao X, Su J, Lonczak A, Northrop LE, Ruiz AA, et al. Tracking embryo implantation using cell-free fetal DNA enriched from maternal circulation at 9 weeks gestation. Mol Hum Reprod 2011;17:434-8.
2. Karnpean R, Tepakhan W, Suankul P, Thingphom S, Poonsawat A, Thanunchaikunlanun N, et al. Genetic background studies of eight common beta thalassemia mutations in Thailand using β-globin gene haplotype and phylogenetic analysis. Genes (Basel) 2022;13:1384.
3. Shang X, Xu X. Update in the genetics of thalassemia: what clinicians need to know. Best Pract Res Clin Obstet Gynaecol 2017;39:3-15.
4. Alwi ZB, Syed-Hassan SNR. Thalassemia in Malaysia. Hemoglobin 2022;46:45-52.
5. Angastiniotis M, Lobitz S. Thalassemias: an overview. Int J Neonatal Screen 2019;5:16.
6. Khaing AA, Myint PP, Paiboonsukwong K, Win N, Fucharoen S, Sripichai O. Clinical severity of β-thalassemia pediatric patients in Myanmar. Hemoglobin 2022;46:66-70.
7. Aliyeva G, Asadov C, Mammadova T, Gafarova S, Abdulalimov E. Thalassemia in the laboratory: pearls, pitfalls, and promises. Clin Chem Lab Med 2018;57:165-74.
8. Ali S, Mumtaz S, Shakir HA, Khan M, Tahir HM, Mumtaz S, et al. Current status of beta‐thalassemia and its treatment strategies. Mol Genet Genomic Med 2021;9:e1788.
9. Winichagoon P, Kumbunlue R, Sirankapracha P, Boonmongkol P, Fucharoen S. Discrimination of various thalassemia syndromes and iron deficiency and utilization of reticulocyte measurements in monitoring response to iron therapy. Blood Cells Mol Dis 2015;54:336-41.
10. Almomani AA, Ala’a SS, Atoom AM, Abdel MBA, Alhmoud JF. Evaluation of the validity of the pre-marriage mean corpuscular volume value as a predictive test for b-thalassemia carrier status. J Med Biochem 2023;42:195-201.
11. Grosso M, Sessa R, Puzone S, Storino MR, Izzo P. Molecular basis of thalassemia. Anemia 2012;2012:341580.
12. Rahimi Z, Muniz A, Parsian A. Detection of responsible mutations for beta thalassemia in the Kermanshah Province of Iran using PCR-based techniques. Mol Biol Rep 2010;37:149-54.
13. Jaafari Z, Sadidi N, Abdolahinia Z, Shahesmaeili A. Prevalence of depression among Iranian patients with beta-thalassemia major: a systematic review and meta-analysis. Iran J Med Sci 2022;47:15-24.
14. El-Fadaly N, Abd-Elhameed A, Abd-Elbar E, El-Shanshory M. Accuracy of reverse dot-blot PCR in detection of different β-globin gene mutations. Indian J Hematol Blood Transfus 2016;32:239-43.
15. Mahdieh N, Rabbani B. Beta thalassemia in 31,734 cases with HBB gene mutations: pathogenic and structural analysis of the common mutations; Iran as the crossroads of the Middle East. Blood Rev 2016;30:493-508.
16. Banan M, Bayat H, Azarkeivan A, Mohammadparast S, Kamali K, Farashi S, et al. The Xmn I and BCL11A single nucleotide polymorphisms may help predict hydroxyurea response in Iranian β-thalassemia patients. Hemoglobin 2012;36:371-80.
17. Epstein CJ, Golbus MS. Prenatal diagnosis of genetic diseases. Am Sci 1977;65:703-11.
18. Wilson RD, Gagnon A, Audibert F, Campagnolo C, Carroll J, Brock JA, et al. Prenatal diagnosis procedures and techniques to obtain a diagnostic fetal specimen or tissue: maternal and fetal risks and benefits. J Obstet Gynaecol Can 2015;37:656-68.
19. Sherlock JK. Minimally invasive prenatal diagnosis of inherited disorders employing trophoblastic cells shed into the endocervical canal [dissertation]. London: University College London; 1999.
20. Monni G, Zoppi MA, Iuculano A, Piras A, Arras M. Invasive or non-invasive prenatal genetic diagnosis? J Perinat Med 2014;42:545-8.
21. Shekhawat DS, Sharma C, Singh K, Singh P, Bhardwaj A, Patwa P. Critical appraisal of droplet digital polymerase chain reaction application for noninvasive prenatal testing. Congenit Anom (Kyoto) 2022;62:188-97.
22. Hanson B, Paternoster B, Povarnitsyn N, Scotchman E, Chitty L, Chandler N. Non-invasive prenatal diagnosis (NIPD): current and emerging technologies. Extracell Vesicles Circ Nucleic Acids 2023;4:3-17.
23. Zafari M, Gill P, Kowsaryan M, Alipour A, Banihashemi A. High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers. J Matern Fetal Neonatal Med 2016;29:3323-8.
24. Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: A review. Hum Mutat 2001;17:439-74.
25. Prajantasen T, Fucharoen S, Fucharoen G, Siriratmanawong N, Pinmuang-Ngam C. Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography. Mol Biol Rep 2013;40:3173-9.
26. Yenilmez ED, Tuli A, Evrüke İC. Noninvasive prenatal diagnosis experience in the Çukurova Region of Southern Turkey: detecting paternal mutations of sickle cell anemia and β-thalassemia in cell-free fetal DNA using high-resolution melting analysis. Prenat Diagn 2013;33:1054-62.
27. Tungwiwat W, Fucharoen G, Fucharoen S, Ratanasiri T, Sanchaisuriya K, Sae-Ung N. Application of maternal plasma DNA analysis for noninvasive prenatal diagnosis of Hb E-β-thalassemia. Transl Res 2007;150:319-25.
28. Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield. PLoS One 2011;6:e25202.
29. Raymond FL, Whittaker J, Jenkins L, Lench N, Chitty LS. Molecular prenatal diagnosis: the impact of modern technologies. Prenat Diagn 2010;30:674-81.
30. Chiu RW, Chan KA, Gao Y, Lau VY, Zheng W, Leung TY, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A 2008;105:20458-63.
31. Treff NR, Tao X, Su J, Lonczak A, Northrop LE, Ruiz AA, et al. Tracking embryo implantation using cell-free fetal DNA enriched from maternal circulation at 9 weeks gestation. Mol Hum Reprod 2011;17:434-8.
| Files | ||
| Issue | Vol 64 No 9 (2026) | |
| Section | Original Articles | |
| Keywords | ||
| IVS-II-1 (G>A) Cell-free fetal DNA Prenatal diagnosis Noninvasive prenatal testing PCR-DHPLC Beta-thalassemia | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Zafari M, Sadeghi T, Banihashemi S, Hashemi A, Bakhtiyari M, Kowsaryan M, Gill P, Musavi H. PCR-DHPLC for Non-Invasive Prenatal Evaluation of the IVS-II-1 (G>A) Mutation in Fetal DNA of β-Thalassemia Major: A Pilot Diagnostic Performance Study. Acta Med Iran. 2026;64(9):594-603.
