Nanoplate Digital PCR for Identification of α0-Thalassemia (SEA Deletion): Carrier Screening and Possible Application to Prenatal Diagnosis

Vollständiger Abstract

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The Southeast Asian deletion (--SEA), the most prevalent α0-thalassemia mutation in Southeast Asia, is a major target of regional thalassemia prevention programs. Coinheritance of α0-thalassemia with other hemoglobinopathies, particularly hemoglobin E (Hb E), complicates routine hematological screening and requires reliable molecular confirmation. This study developed and validated a duplex nanoplate digital polymerase chain reaction (dPCR) assay for simultaneous identification of the wild-type α-globin and --SEA deletion alleles in a single reaction. The assay was established on the QIAcuity nanoplate platform and evaluated using 171 blinded leftover DNA specimens from individuals with Hb E. Diagnostic performance was compared with conventional gap-PCR. Distinct fluorescence amplitude patterns enabled discrimination among normal, heterozygous, and homozygous --SEA genotypes in a single closed tube. Among the 171 specimens, 70 were identified as --SEA carriers and 101 as non-carriers, showing 100% concordance with gap-PCR. All subjects with Hb E + A2 levels ≥ 25% were negative for the --SEA deletion, whereas 56.0% of those with levels < 25% carried the deletion. In the representative prenatal case, duplex dPCR accurately identified parental carrier status and a homozygous --SEA fetus. This assay provides an accurate and practical molecular confirmatory assay for thalassemia screening and prenatal diagnosis in Southeast Asia.