title: Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR.
abstract: BACKGROUND
  Fetal-maternal ABO incompatibility is a frequent cause of hemolytic disease of the fetus and newborn (HDFN). The routine serological testing of maternal IgG antibody level to predict HDFN shows low reliability. Non-invasive fetal ABO genotyping could provide a new avenue for predicting ABO-HDFN in early pregnancy. The aim of our study is to investigate the feasibility of fetal ABO genotyping in maternal plasma with real-time PCR.
  
  
  METHODS
  Plasma samples were collected from a total of 73 blood group O pregnant women between 12 and 25 weeks of gestation, and then DNA was extracted from the maternal plasma containing cell-free fetal DNA (cffDNA). TaqMan-based real-time PCR was performed after methylation-sensitive restriction enzyme to detect hypermethylated RASSF1A sequences of fetal origin in maternal plasma. Fetal ABO genotypes were determined by SYBR-based real-time PCR with allele-specific primers. The performance of the fetal ABO genotyping was assessed by the blood group serology results of the newborns.
  
  
  RESULTS
  The fetal RASSF1A sequences were detectable in all the 73 plasma samples, which confirmed the successful extraction of cffDNA. The diagnostic accuracy of fetal ABO genotyping was 93.2%, in which the accuracy of fetal genotype OO, OA and OB was 100%, 83.3% and 96.8%, respectively.
  
  
  CONCLUSIONS
  We have developed a rapid and reliable protocol for fetal ABO genotyping in maternal plasma using real-time PCR. This protocol is suitable for routine prenatal diagnose of HDFN and forensic analysis.
authors.forename: Wenqian
authors.initials: W
authors.lastname: Song
authors.forename: Shihang
authors.initials: S
authors.lastname: Zhou
authors.forename: Linnan
authors.initials: L
authors.lastname: Shao
authors.forename: Ni
authors.initials: N
authors.lastname: Wang
authors.forename: Lingzi
authors.initials: L
authors.lastname: Pan
authors.forename: Weijian
authors.initials: W
authors.lastname: Yu
mesh_terms: D000017:ABO Blood-Group System / Q000097:blood* / Q000235:genetics*; D000328:Adult; D052160:Benzothiazoles; D003959:Diamines; D005260:Female; D005312:Fetal Blood / Q000378:metabolism; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry; D011247:Pregnancy / Q000097:blood*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000235:genetics; D055815:Young Adult
chemical_list: D000017:ABO Blood-Group System; D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; C098022:SYBR Green I