Noninvasive fetal genotyping of human platelet antigen‐1a using targeted massively parallel sequencing. Issue 6 (15th April 2015) - Wienzek‐Lischka, Sandra
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. STUDY DESIGN AND 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. CONCLUSION: 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.