Wienzek-Lischka Sandra, Krautwurst Annika, Fröhner Vanessa, Hackstein Holger, Gattenlöhner Stefan, Bräuninger Andreas, Axt-Fliedner Roland, Degenhardt Jan, Deisting Christina, Santoso Sentot, Sachs Ulrich J, Bein Gregor
Institute for Clinical Immunology and Transfusion Medicine.
Institute of Pathology.
Transfusion. 2015 Jun;55(6 Pt 2):1538-44. doi: 10.1111/trf.13102. Epub 2015 Apr 15.
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.
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.
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.
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.
对于有胎儿和新生儿同种免疫性血小板减少症（FNAIT）病史的孕妇，需要进行胎儿人类血小板抗原（HPA）基因分型，以确定胎儿是否有风险以及是否需要采取产前干预措施来预防胎儿出血。最近公布了利用母体血浆游离DNA对HPA等位基因进行无创基因分型的方法，但确实缺乏内部对照以排除假阴性结果。
从4名有抗HPA-1a导致的FNAIT病史的孕妇及对照者的血浆中分离游离DNA。设计了一组引物，靶向整合素β3（ITGB3，HPA-1）、整合素α2β（ITGA2B，HPA-3）、整合素α2（ITGA2，HPA-5）、CD109（HPA-15）、RHD、RHCE、KEL、DARC、溶质载体家族14成员1（SLC14A1）、血型糖蛋白A（GYPA）、血型糖蛋白B（GYPB）和Y染色体性别决定区（SRY）的单核苷酸多态性（SNP）/外显子区域侧翼序列。通过半导体技术对这些区域和8个匿名SNP进行大规模平行测序。
靶向SNP的平均（±标准差）读数为5255（±2838）。在中位数为4.5（范围2 - 8）个多态性位点检测到胎儿DNA。母体血浆游离DNA中胎儿DNA的平均分数浓度为8.36%（范围4.79% - 15.9%）。对于HPA-1，在所有HPA-1ab胎儿中均检测到非母体ITGB3序列（c.176T，HPA-1a）。明确鉴定出1例HPA-1bb胎儿，表明该妊娠不存在FNAIT风险。
我们已成功建立大规模平行测序作为一种新型可靠的方法，用于胎儿HPA-1a等位基因的无创基因分型。该技术还可能有助于安全检测其他经常涉及FNAIT和新生儿溶血病的胎儿血型多态性。
