title: Microsatellite markers within --SEA breakpoints for prenatal diagnosis of HbBarts hydrops fetalis.
abstract: 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.
authors.affiliation: Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Sherry Sze Yee
authors.initials: SS
authors.lastname: Ho
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Chong
authors.forename: Evelyn S C
authors.initials: ES
authors.lastname: Koay
authors.forename: Yiong Huak
authors.initials: YH
authors.lastname: Chan
authors.forename: Ponnusamy
authors.initials: P
authors.lastname: Sukumar
authors.forename: Lily-Lily
authors.initials: LL
authors.lastname: Chiu
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.forename: Ashim
authors.initials: A
authors.lastname: Roy
authors.forename: Mary
authors.initials: M
authors.lastname: Rauff
authors.forename: Lin Lin
authors.initials: LL
authors.lastname: Su
authors.forename: Arijit
authors.initials: A
authors.lastname: Biswas
authors.forename: Mahesh
authors.initials: M
authors.lastname: Choolani
mesh_terms: 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*
chemical_list: D006455:Hemoglobins, Abnormal; D005914:Globins; D004247:DNA; C030448:hemoglobin Bart's