Amplicon Sequencing-Based Noninvasive Fetal Genotyping for RHD-Positive D Antigen-Negative Alleles
Amplicon Sequencing-Based Noninvasive Fetal Genotyping for RHD-Positive D Antigen-Negative Alleles
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DOI：
10.1373/clinchem.2019.307074
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发表时间：
2019-10-01
影响因子：
9.3
通讯作者：
Hata, Kenichiro
中科院分区：
文献类型：
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作者：
Takahashi, Ken;Migita, Ohsuke;Hata, Kenichiro
背景技术背景：为了避免胎儿和新生儿的溶血性疾病所造成的母体同种抗体对胎儿Rh抗原，抗D免疫球蛋白是常规管理RhD阴性孕妇在日本。胎儿RHD基因分型使用无细胞DNA可以防止不必要的抗体管理，但是，目前基于PCR的方法，检测RHD缺失，不解决RHD阳性D抗原阴性等位基因在非白人populationwithout additional investigations.METHODS：我们开发了一种扩增子测序方法，可以估计类型的父系遗传的胎儿RHD等位基因从4个主要的RHD等位基因在日本人口：D抗原阳性等位基因（RHD*01，92.9%）和3D抗原阴性等位基因（RHD*01N.01，6.6%; RHD*01EL.01，0.3%; RHD* 01N.04，0.1%），使用从孕妇血浆获得的游离DNA。即使RhD阴性孕妇具有RHD阳性D抗原阴性等位基因，该方法也能正确确定胎儿RhD类型：结论：该方法是一种可靠的无创性RHD基因分型方法。使用相同的引物对扩增2个不同的区域，并在随后的作图过程中通过它们的序列差异来区分它们的基因分型原理在理论上也适用于非洲人中流行的RHD阳性D抗原阴性等位基因。因此，这种方法提供了一个机会，可以考虑向东亚和非洲国家的RhD阴性孕妇靶向给予抗D免疫球蛋白，并提高几个欧洲国家在全国范围内实施的胎儿RHD基因分型的特异性。(C)2019年美国临床化学协会
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