Microsatellite Markers within SEA Breakp

We sought to develop a rapid prenatal diagnostic test for simultaneous detection of HbBarts hydrop fetalis and exclusion of maternal contamination. We developed a multiplex quantitative fluorescent PCR (QF-PCR) test that detects the presence/absence of 2 microsatellite markers (16PTEL05/16PTEL06) located within breakpoints of the Southeast Asia (—SEA) deletion. HbBarts hydrop fetalis (—SEA—SEA) is diagnosed by absence of both markers, and maternal contamination of fetal DNA is excluded by absence of noninherited maternal alleles. Fetal and parental DNA samples from 50 families were analyzed in a blinded clinical validation study, and QF-PCR results were compared with their respective molecular genotypes. The multiplexer QF-PCR results included correct diagnoses of HbBarts hydrop fetalis in 11 of the fetuses tested, correct verification as unaffected in 20 fetuses, and correct identification as either carriers (αα—SEA) or unaffected homozygotes in 18. Sensitivity for diagnosis of HbBarts hydrop fetalis was 100%, and specificity was 100%. None of the samples tested showed traces of noninherited maternal alleles; false-positives due to maternal contamination were eliminated. This novel strategy using cell-free fetal DNA in maternal plasma could form the basis for noninvasive testing for HbBarts hydrop fetalis.