Amplicon Sequencing-Based Noninvasive Fetal Genotyping for RHD-Positive D Antigen-Negative Alleles

Abstract

BACKGROUND To avoid hemolytic disease of the fetus and newborn resulting from maternal alloantibodies against fetal Rh antigens, anti-D immunoglobulin is routinely administered to RhD-negative pregnant women in Japan. Fetal RHD genotyping using cell-free DNA may prevent unnecessary antibody administration; however, current PCR-based methods, which detect RHD deletion, do not address the higher rates of RHD-positive D antigen-negative alleles in nonwhite populations without additional inspections. METHODS We developed an amplicon-sequencing method that could estimate the type of paternally inherited fetal RHD allele from 4 major RHD alleles in the Japanese population: the D antigen-positive allele (RHD*01, 92.9%) and 3 D antigen-negative alleles (RHD*01N.01, 6.6%; RHD*01EL.01, 0.3%; RHD*01N.04, 0.1%) using cell-free DNA obtained from the blood plasma of pregnant women. RESULTS The method correctly determined the fetal RhD type even when RhD-negative pregnant women possessed an RHD-positive D antigen-negative allele: RHD*01EL.01 or RHD*01N.04. CONCLUSIONS This method is a reliable noninvasive fetal RHD genotyping method for Japanese and other East Asian populations. The genotyping principle of amplifying 2 different regions using the same primer pair and distinguishing them by their sequence difference during the subsequent mapping procedure is also theoretically applicable to RHD-positive D antigen-negative alleles prevalent in Africans. Therefore, this method offers an opportunity to consider targeted administration of anti-D immunoglobulin to RhD-negative pregnant women in East Asian and African countries and to increase the specificity of the fetal RHD genotyping implemented nationwide in several European countries.

Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma

“…For the populations in which several variants are responsible for RhD-negative status, other methods should be considered, for instance, the NGS (Next Generation Sequencing) method [ 57 ].…”

Insufficient gestational weight gain associated with higher DNA methylation in cord blood cells

“…PCR was performed by TaKaRa Ex Taq® (TaKaRa, Shiga, Japan). Deep amplicon sequencing was performed as described previously [53]. The obtained amplicons for 5 targeted regions from the same subject's sample were pooled and subjected to adaptor ligation and indexing (6 cycles) using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA).…”

Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles

“…We have recently established an amplicon-based noninvasive fetal genotyping method that distinguishes the wild-type RHD allele not only from the RHD- negative D antigen-negative allele (the RHD deletion allele), but also from RHD -positive D antigen-negative alleles [ 4 ]. This method requires PCR amplification from four genomic intervals, upstream and downstream Rhesus boxes , RHD exon 9, and RHCE exon 9.…”

“…The 148-bp PCR products contain two base differences that distinguish two genes, and also cover the point mutation site in exon 9 of the RHD*01EL.01 allele (c.1227G/A) that distinguishes it from the wild-type allele ( RHD*01 ). Although two regions are co-amplified with one primer pair, attachment of adaptor sequences to the PCR amplicons followed by NGS allowed us to accurately map each of the co-amplified sequences to its origin because of the one or two base differences between two regions, in the data analysis procedure [ 4 ].

Fig.

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“…Lower and upper marker peaks are present at the positions of 35 bp and 10,380 bp, respectively. In our conventional [ 4 ] and one-step PCR protocols ( C , F ), multiplex PCR using two pairs of primers gave rise to specific amplification of target genomic intervals ( D , G ). K A data analysis workflow showing the informatics tools used and their step-by-step functions.…”