Next-generation sequencing of 35 RHD variants in 16 253 serologically D− pregnant women in the Finnish population

Abstract

Fetal RHD screening for targeted routine antenatal anti-D prophylaxis has been implemented in many countries, including Finland, since the 2010s. Comprehensive knowledge of the RHD polymorphism in the population is essential for the performance and safety of the anti-D prophylaxis program. During the first 3 years of the national screening program in Finland, over 16 000 samples from RhD− women were screened for fetal RHD; among them, 79 samples (0.5%) containing a maternal variant allele were detected. Of the detected maternal variants, 35 cases remained inconclusive using the traditional genotyping methods and required further analysis by next-generation sequencing (NGS) of the whole RHD gene to uncover the variant allele. In addition to the 13 RHD variants that have been previously reported in different populations, 8 novel variants were also detected, indicating that there is more variation of RHD in the RhD− Finnish population than has been previously known. Three of the novel alleles were identified in multiple samples; thus, they are likely specific to the original Finnish population. National screening has thus provided new information about the diversity of RHD variants in the Finnish population. The results show that NGS is a powerful method for genotyping the highly polymorphic RHD gene compared with traditional methods that rely on the detection of specific nucleotides by polymerase chain reaction amplification.

RhD alloimmunization by DEL variant missed in donor testing

“…11,23 Currently used methods in RHD genotyping, PSR-SSP, and Sanger sequencing are insufficient to identify many variant alleles, especially intronic variations. 30 We suggest additional gene investigation not covered in this study including yet unexplored regions of regulation and intron regions to justify our serological finding.…”

“…Furthermore, it is hard to quantify the number of antigen sites able to elicit the immune response, but studies showed it can be as low as 30 D antigen sites 11,23 . Currently used methods in RHD genotyping, PSR‐SSP, and Sanger sequencing are insufficient to identify many variant alleles, especially intronic variations 30 . We suggest additional gene investigation not covered in this study including yet unexplored regions of regulation and intron regions to justify our serological finding.…”

Application of Blood Group Genotyping by Next-Generation Sequencing in Various Immunohaematology Cases

“…Such a limitation can be overcome by short-read sequencing based on PCR enrichment, discriminating between homologous regions or long-read sequencing such as nanopore sequencing. A short-read sequencing method based on long-range PCR enrichment, specifically amplifying the RHD gene, has already been described [18, 32].…”

Apparent changes in RhD status during pregnancy: A case study of weak D variant due to RHD*DAU‐2

“…Transfusing blood donation from these individuals to RhD negative recipients can risk formation of anti-D. Consequently, many countries routinely use genotyping to identify RHD variants in donors and recipients. [5][6][7] The management of pregnancies of women with variant RhD phenotypes is complicated by the fact that routine cff DNA testing cannot accurately predict the RhD phenotype of the fetus due to the presence of a maternal RHD gene. The fetus may express epitopes of RhD that the mother lacks, causing alloimmunisation and therefore anti-D prophylaxis should be offered.…”