Long read RHD-RHCE sequencing of samples with inconsistent fetal RHD real-time PCR results

According to German guidelines, non-invasive fetal RHD analysis should be offered to every RhD-negative pregnant woman to avoid unnecessary anti-D prophylaxis for women with an RHD negative fetus. The fetal RHD genotype can reliably be determined in maternal blood by real-time PCR (rtPCR) in the vast majority of cases. Long-read RHD-RHCE sequencing was used to resolve discrepancies between RhD phenotype and genotyping.

We use two different rtPCR test systems in parallel for fetal RHD detection in maternal blood: the commercial Free DNA Fetal Kit RhD Duplex Kit (Institut de Biotechnologies Jacques Boy) and an in-house rtPCR method. Both methods utilize amplification of exon 5, 7, and 10 of the RHD gene on the CFX Opus Real-Time PCR System (Biorad). Early amplification (Ct<34.5) of at least one of the RHD exons indicates the presence of maternal RHD sequences. For sequencing, RHD and RHCE are enriched either by long-range PCR, covering the whole genes of RHD and RHCE, or by a customized hybridization-based panel (Twist Bioscience). Long read sequencing is performed on a Sequel IIe (PacBio), and data are analyzed using SeqNext software (JSI).

In 33 out of 2008 samples for fetal RHD genotyping, the presence of maternal RHD sequences was suggested despite negative RhD serology. Nine samples showed early amplification of all exons, ten samples showed amplification of exons 7 and 10, and 14 samples of exon 10 only. Long-read RHD and RHCE sequencing has been completed for 11 samples so far. The presence of maternal RHD sequences was confirmed in all samples. In samples with early exon 5, 7, and 10 amplification, RHD11, RHD15, two RHD01EL.08 alleles, and a novel allele with a splice site mutation were detected. All 3 samples with early exon 7 and 10 amplification were identified as RHD08N.01. Three samples with early exon 10 amplification can be explained by hybrid RHD-CE-D alleles.

In 1.6% of serologically RhD-negative samples, the presence of maternal RHD sequences was detected by rtPCR. Complete long read sequencing of RHD and RHCE genes enabled the determination of the underlying genetic basis for missing RhD expression. Rare genetic variants were detected in our patient population from Germany. Long read sequencing is a powerful tool to assign rare RHD and RHCE alleles, even with complex genetic structures like RHD-RHCE hybrid alleles.

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