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Transfusion. 2015 Jun;55(6 Pt 2):1538-44. doi: 10.1111/trf.13102. Epub 2015 Apr 15.

Noninvasive fetal genotyping of human platelet antigen-1a using targeted massively parallel sequencing.

Author information

1
Institute for Clinical Immunology and Transfusion Medicine.
2
Institute of Pathology.
3
Division of Prenatal Medicine, Department of Obstetrics and Gynecology, Justus-Liebig-University, Giessen, Germany.

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.

PMID:
25873286
DOI:
10.1111/trf.13102
[Indexed for MEDLINE]

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