Diagnosis of alpha-thalassemia-1 Southeast Asian type deletion and fetal gender by single-tube multiplex real-time PCR.

Background: The combination of an x-linked hematologic disorder with a heterozygous alpha-thalassemia-1 Southeast Asian (SEA) type deletion might lead to severe anemia in male infants. This study is to develop a simple and cost-effective single tube multiplex real-time PCR for the diagnosis of alpha-thalassemia-1 SEA type deletion and detect fetal gender.

Methods: Multiplex real-time polymerase chain reaction (PCR) for the detection of alpha-thalassemia-1 SEA type deletion gene, wild type alpha-globin gene, and sex-determining region Y (SRY) gene was validated by analysis of 95 cord blood samples (60 normal individuals, 28 heterozygous of alpha-thalassemia-1 SEA type deletion and 7 Bart's hydrops fetalis). The change in threshold cycle (deltaC(T)) was analyzed by subtracting the C(T-mutant) from C(T-wild) type.

Results: Mean deltaC(T) values were significantly different among these three groups, and a SRY gene determination was 100% in concordance with fetal genders. Furthermore, analysis of fetal gender did not affect the deltaC(T) of alpha-thalassemia-1 SEA detection.

Conclusions: Combined alpha-thalassemia-1 SEA type detection and fetal gender determination in a single-tube multiplex real-time PCR is an alternative assay for a conventional method for the diagnosis of alpha-thalassemia-1 SEA deletion and fetal gender.