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

作者：Ken Takahashi, Ohsuke Migita, A. Sasaki, Michiko Nasu, Akihiro Kawashima, Akihiko Sekizawa, Taisuke Sato, Yuki Ito, Haruhiko Sago, Aikou Okamoto, Kazuhiko Nakabayashi, Kenichiro Hata · 发表于：Clinical Chemistry · 年份：2019 · DOI：10.1373/clinchem.2019.307074 · 被引用次数：19 · 研究领域：Blood groups and transfusion、Prenatal Screening and Diagnostics、Parvovirus B19 Infection Studies

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 ...