Non-invasive fetal HPA genotyping by UMI-NGS: a robust method for antenatal diagnosis including 48 fetal DNA markers

Non-invasive fetal HPA typing is a valuable tool to identify the pregnancies at risk of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Different approaches have been developed, mainly based on real-time PCR and droplet digital-PCR. Those methods have a limited ability to multiplex and require replicates due to the contamination risk. Moreover, in order to exclude false-negative results caused by insufficient cell-free fetal DNA, the presence of fetal DNA is usually assessed with a single epigenetic marker whereas large single-nucleotide variant (SNV) panels are now available to more accurately measure the sample's fetal fraction.

We developed an innovative method for the simultaneous genotyping of HPA-1, -2, -3, -4, -5, -6, -9 and 15, in combination with a panel of 48 SNV markers, based on cell-free DNA target-enrichment with specific probes. An improved accuracy using NGS sequencing was reached with the Unique Molecular Identifiers (UMIs); these molecular barcodes are short sequences used to uniquely tag each molecule in a sample library.

81 plasma samples were collected from French and Spanish pregnant women, from 10 to 40 weeks of gestation ([wg]; 4 samples <12 wg; 38 samples 13-24 wg; 39 samples >24 wg). The panel of 48 SNVs allowed a precise quantification of fetal DNA (range: 1.1%-16.1%). All samples but one gave concordant results with the confirmed HPA genotypes. One sample was discordant for HPA-2 and HPA-3, due to false negative cffDNA results for these two loci. However, a low amount of fetal fraction in that sample was effectively alerted by the SNV markers results.

In our experience, 16 samples can be simultaneously sequenced and analyzed in a 72 hours assay. UMIs NGS sequencing of HPA and SNV markers constitutes a robust and sensitive method for non-invasive fetal HPA genotyping.