Noninvasive fetal RHD genotyping by microfluidics digital PCR using maternal plasma from two alloimmunized women with the variant RHD(IVS3+1G>A) allele

Abstract

The noninvasive prenatal determination of fetal RhD blood group can be achieved by analyzing cell-free fetal DNA in maternal plasma. An RhD-positive fetus can be identified by detecting the presence of RHD gene sequences in the plasma of an RhD-negative woman who does not possess the gene. 1Many research groups have demonstrated the high diagnostic accuracy of this approach, and the test has now been available as a clinical service in many laboratories in Europe and the USA.In addition to the complete deletion of the RHD gene, a number of RHD gene variants that consist of single-nucleotide mutations have been reported to be associated with RhDnegative or the DEL phenotype, that is, a very weak expression of the D antigen. Pregnant women who harbor RHD variants, such as ones with the RHD(IVS3+1G>A) mutation, are capable of producing anti-D alloantibodies and thus are at risk for hemolytic disease of the fetus and newborn (HDFN).2,3 Thus far, noninvasive prenatal RHD genotyping for women carrying such intact but dysfunctional RHD variants has not been reported. The challenge is due to the high background of maternal DNA in maternal plasma and fetal DNA levels amount to only 10% of the total plasma DNA molecules. 4 For pregnant women with a complete RHD gene deletion, the fetal RHD DNA detection is free from maternal background interference because the whole gene is absent in the maternal genome. On the other hand, for maternal plasma involving maternal RHD variants, one has to discriminate the fetal-allele sequences among the high background of maternal-allele sequences that differ from each other by a single nucleotide only. Digital polymerase chain reaction (PCR) has been shown to be a sensitive and specific tool for detecting single nucleotide variations between the mother and the fetus in maternal plasma. 5 In this study, we investigated the feasibility of using digital PCR to determine the fetal inheritance of RHD allele noninvasively using maternal plasma. As proof-of-principle, we investigated two alloimmunized pregnant women carrying the RHD(IVS3+1G>A) mutation. 3This investigation was triggered after two separate obstetric cases that were both Caucasian, phenotyped as RhD-negative by hospital serology, and were sensitized to the D antigen. Maternal plasma samples were referred to the blood service for noninvasive prenatal RHD genotyping. However, the fetal RHD typing by real-time PCR was unsuccessful due to the presence of maternal RHD allele sequences in maternal plasma. The RHD dosage analysis of maternal genomic DNA by PCR 6 showed that the two women were hemizygous for RHD, and they carried the DEL associated RHD*IVS3+1G>A allele as detected using a commercial single nucleotide polymorphism (SNP) based blood group genotyping platform (BLOODchip micorarray, Progenika Biopharma S.A.). The two women were confirmed serologically to express the DEL

Quantitative Analysis of Pork and Chicken Products by Droplet Digital PCR

“…As a result, dPCR has been applied in a wide range of areas including quantitative gene expression analysis [12], single nucleotide polymorphism analysis [13], genotyping [14], rare variant and copy number variation detection, pathogen detection [15], drug resistance research [16], and noncoding RNA research [17]. …”

14 Years of Polish Experience in Non-Invasive Prenatal Blood Group Diagnosis

“…Different scientific teams report different cffDNA quantities depending on the method of determination. They vary from 3-6% using qrtPCR to 10% using digital PCR [6,22]. In our protocols, it is obligatory to estimate the level of plasma DNA by amplification of the CCR5 gene in order to exclude samples with an excessive background of genomic DNA released from blood cells.…”

Applicability of digital PCR to the investigation of pediatric-onset genetic disorders

“…dPCR can detect paternally-inherited, fetal RHD in maternal plasma [34], [45]. dPCR can accurately detect maternal RHD variants like RHD(IVS3   +   1G   >   A) in the presence of cffDNA containing paternally-inherited RHD [46].…”