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

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. (C) 2019 American Association for Clinical Chemistry