Microsatellite markers within --SEA breakpoints for prenatal diagnosis of HbBarts hydrops fetalis.
BACKGROUND
We sought to develop a rapid prenatal diagnostic test for simultaneous detection of HbBarts hydrops fetalis and exclusion of maternal contamination.


METHODS
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 hydrops 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.


RESULTS
The multiplex QF-PCR results included correct diagnoses of HbBarts hydrops fetalis in 11 of the fetuses tested, correct verification as unaffected in 20 fetuses, and correct identification as either carriers (alphaalpha/(-SEA)) or unaffected homozygotes in 18. Misidentification as unaffected occurred for 1 carrier. Sensitivity for diagnosis of HbBarts hydrops fetalis was 100% [lower 95% confidence interval, 76.2%], and specificity was 100% (lower 95% confidence interval, 92.6%). None of the samples tested showed any traces of noninherited maternal alleles; thus false-positives because of maternal contamination were eliminated.


CONCLUSIONS
In this QF-PCR method, detection of maternally and paternally inherited fetal alleles allowed diagnosis of the double-deletion syndrome, and the ability to differentiate between these alleles allowed simultaneous exclusion of maternal contamination of the fetal genetic material. This novel strategy using cell-free fetal DNA in maternal plasma could form the basis for noninvasive testing for HbBarts hydrops fetalis.
Ho
Sherry Sze Yee
SS
Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Chong
Samuel S
SS
Koay
Evelyn S C
ES
Chan
Yiong Huak
YH
Sukumar
Ponnusamy
P
Chiu
Lily-Lily
LL
Wang
Wen
W
Roy
Ashim
A
Rauff
Mary
M
Su
Lin Lin
LL
Biswas
Arijit
A
Choolani
Mahesh
M
D000483:Alleles; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005260:Female; D005333:Fetus; D005914:Globins / Q000235:genetics*; D006455:Hemoglobins, Abnormal; D006579:Heterozygote; D006801:Humans; D015160:Hydrops Fetalis / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008431:Maternal-Fetal Exchange; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*
D006455:Hemoglobins, Abnormal; D005914:Globins; D004247:DNA; C030448:hemoglobin Bart's