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

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.