title: Noninvasive fetal genotyping of human platelet antigen-1a using targeted massively parallel sequencing.
abstract: BACKGROUND
  Fetal human platelet antigen (HPA) genotyping is required to determine whether the fetus is at risk and whether prenatal interventions to prevent fetal bleeding are required in pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Methods for noninvasive genotyping of HPA alleles with the use of maternal plasma cell-free DNA were published recently but do lack internal controls to exclude false-negative results.
  
  
  METHODS
  Cell-free DNA was isolated from plasma of four pregnant women with a history of FNAIT caused by anti-HPA-1a and controls. A primer panel was designed to target sequences flanking single-nucleotide polymorphisms (SNPs)/exonic regions of ITGB3 (HPA-1), ITGA2B (HPA-3), ITGA2 (HPA-5), CD109 (HPA-15), RHD, RHCE, KEL, DARC, SLC14A1, GYPA, GYPB, and SRY. These regions and eight anonymous SNPs were massively parallel sequenced by semiconductor technology.
  
  
  RESULTS
  The mean (±SD) number of reads for targeted SNPs was 5255 (±2838). Fetal DNA was detected at a median of 4.5 (range, 2-8) polymorphic loci. The mean fractional fetal DNA concentration in cell-free maternal plasma was 8.36% (range, 4.79%-15.9%). For HPA-1, nonmaternal ITGB3 sequences (c.176T, HPA-1a) were detected in all HPA-1ab fetuses. One HPA-1bb fetus was unequivocally identified, showing the pregnancy was not at risk of FNAIT.
  
  
  CONCLUSIONS
  We have successfully established massively parallel sequencing as a novel reliable method for noninvasive genotyping of fetal HPA-1a alleles. This technique may also allow the safe detection of other fetal blood group polymorphisms frequently involved in FNAIT and hemolytic disease of the newborn.
authors: Institute for Clinical Immunology and Transfusion Medicine.
authors: Sandra
authors: S
authors: Wienzek-Lischka
authors: Institute for Clinical Immunology and Transfusion Medicine.
authors: Annika
authors: A
authors: Krautwurst
authors: Institute for Clinical Immunology and Transfusion Medicine.
authors: Vanessa
authors: V
authors: Fröhner
authors: Institute for Clinical Immunology and Transfusion Medicine.
authors: Holger
authors: H
authors: Hackstein
authors: Institute of Pathology.
authors: Stefan
authors: S
authors: Gattenlöhner
authors: Institute of Pathology.
authors: Andreas
authors: A
authors: Bräuninger
authors: Division of Prenatal Medicine, Department of Obstetrics and Gynecology, Justus-Liebig-University, Giessen, Germany.
authors: Roland
authors: R
authors: Axt-Fliedner
authors: Division of Prenatal Medicine, Department of Obstetrics and Gynecology, Justus-Liebig-University, Giessen, Germany.
authors: Jan
authors: J
authors: Degenhardt
authors: Division of Prenatal Medicine, Department of Obstetrics and Gynecology, Justus-Liebig-University, Giessen, Germany.
authors: Christina
authors: C
authors: Deisting
authors: Institute for Clinical Immunology and Transfusion Medicine.
authors: Sentot
authors: S
authors: Santoso
authors: Institute for Clinical Immunology and Transfusion Medicine.
authors: Ulrich J
authors: UJ
authors: Sachs
authors: Institute for Clinical Immunology and Transfusion Medicine.
authors: Gregor
authors: G
authors: Bein
mesh_terms: D016824:Antigens, Human Platelet / Q000235:genetics*; D001774:Blood Chemical Analysis / Q000379:methods; D016022:Case-Control Studies; D002474:Cell-Free System; D005260:Female; D005315:Fetal Diseases / Q000175:diagnosis* / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007231:Infant, Newborn; D039661:Integrin beta3; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy / Q000097:blood; D011296:Prenatal Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000175:diagnosis* / Q000235:genetics
chemical_list: D016824:Antigens, Human Platelet; C061248:ITGB3 protein, human; D039661:Integrin beta3