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Enhancing Hemoglobin Bart's hydrops fetalis syndrome prevention: a single-tube multiplex real-time PCR assay for the comprehensive detection of four significant α0-thalassemia deletions (--SEA, --THAI, --CR, and --SA) found in Thailand.

Rossarin Karnpean·Sanita Singsanan·Kasama Wongprachum·Punchita Rujirachaivej·Rawiporn Tiyasirichokchai·Prapaporn Panichchob·Paweesuda Rattanakoch·Praew Phermthong·Wittaya Jomoui
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Abstract

BACKGROUND: Hemoglobin (Hb) Bart's hydrops fetalis is a major public health concern in Southeast Asia, particularly in Thailand. Current screening strategies target the two most common α0 -thalassemia deletions (--SEA and --THAI). METHOD: In this study, we developed a single-tube multiplex real-time PCR assay for the simultaneous detection of four clinically relevant α0-thalassemia deletions (--SEA, --THAI, --CR, and --SA). The assay was validated using 538 clinical samples with diverse thalassemia genotypes and compared against conventional gap-PCR as the reference method. Analytical performance, including sensitivity, specificity, and limit of detection (LOD), was evaluated. In addition, clinical utility was assessed in 22 prenatal diagnosis cases at risk of Hb Bart's hydrops fetalis. RESULTS: The study cohort demonstrated substantial genetic heterogeneity, comprising 43 distinct genotypes. The developed assay achieved 100% sensitivity and specificity for all targeted deletions, with complete concordance with gap-PCR results. No cross-reactivity was observed with α+-thalassemia. The assay demonstrated a high analytical sensitivity with a LOD of 9.76 × 10-3 ng per reaction. Whereas in prenatal diagnosis, all 22 fetal genotypes were accurately identified, including five cases of homozygous --SEA and one rare compound heterozygous --SEA/--CR fetus. CONCLUSIONS: This study presents a rapid, accurate, and cost-effective multiplex real-time PCR assay capable of detecting both common and rare α0-thalassemia deletions in a single reaction. The assay demonstrates strong potential for implementation in routine clinical laboratories and large-scale population screening, contributing to improved prevention and control of severe thalassemia syndromes in high-prevalence regions.

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HumansHydrops FetalisThailandHemoglobins, Abnormalalpha-ThalassemiaFemalePrenatal DiagnosisReal-Time Polymerase Chain ReactionSensitivity and SpecificityPregnancyMultiplex Polymerase Chain ReactionGenotypeSequence DeletionGene DeletionChiang Rai deletionHb Bart’s hydrops fetalisSEA deletionSouth Africa deletionThai deletiondiseasereal-time PCRα0-thalassemiaJournal ArticleResearch Support, Non-U.S. Gov't

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Rossarin Karnpean, Sanita Singsanan, Kasama Wongprachum, Punchita Rujirachaivej, Rawiporn Tiyasirichokchai, Prapaporn Panichchob, Paweesuda Rattanakoch, Praew Phermthong, Wittaya Jomoui. 2026-07-01. Enhancing Hemoglobin Bart's hydrops fetalis syndrome prevention: a single-tube multiplex real-time PCR assay for the comprehensive detection of four significant α0-thalassemia deletions (--SEA, --THAI, --CR, and --SA) found in Thailand.. https://doi.org/10.1080/07853890.2026.2690745

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Disease subjectFemale subjectGenotype subjectHumans subjectJournal Article subjectJournal Article; Research Support, Non-U.S. Gov't resourcePregnancy subjectPrenatal Diagnosis subject

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