Apply Microsatellite markers in Prenatal Diagnosis of Hb Bart's Hydrops Fetalis in Vietnamese

Abstract

Hemoglobin (Hb) Bart’s hydrops fetalis is a fatal condition associated with homozygous for --SEA deletion. Prenatal diagnosis of the disease is usually done by gap-PCR. However, misdiagnosis can occur with allelic dropout and maternal contamination. Diagnosis using more than one method is preferred. We sought to develop a rapid prenatal diagnostic test for simultaneous detection of Hb Bart’s hydrops fetalis and exclusion of maternal contamination using a multiplex quantitative fluorescent PCR test that amplify of 2 microsatellite markers (16PTEL05/16PTEL06) located within breakpoints of the Southeast Asia (--SEA) deletion and a marker (D16S539) outside this breakpoint as control. Hb Bart’s hydrops fetalis (--SEA/--SEA) is diagnosed by absence of both markers 16PTEL05 and 16PTEL06 with the presence of D16S539, and maternal contamination of fetal DNA is excluded by absence of non-inherited maternal alleles. Fetal and parental DNA samples from 11 families were analyzed by this multiplex QF-PCR, and QF-PCR results were compared with their respective molecular genotypes to validate this method. The multiplex QF-PCR resulted in correct diagnosis of 1 sample with HbBarts hydrops fetalis, 13 samples as either α-thalassemia carriers (αα/--SEA) or unaffected homozygotes (αα/αα), and the remaining 19 samples as unaffected. Multiplex QF-PCR for that amplifies three markers 16PTEL05, 16PTEL06 and D16S539 was developed successfully. In this QF-PCR method, detection of maternally and paternally inherited alleles of these microsatellite marker in fetal DNA samples allowed diagnosis of Hb Bart’s hydrops fetalis, and the ability to differentiate between these alleles allowed simultaneous exclusion of maternal contamination these fetal DNA samples. Key words: Hb Bart’s hydrops fetalis, quantitative fluorescent PCR, microsatellite markers