Study on RHD and RHCE gene mRNA based on three generation sequencing technology

Objective To establish RH gene mRNA sequencing method based on nanopores sequencing and to explore the RHD and RHCE mRNA transcripts in D positive and Del individuals. Methods From March 2021 to May 2022, 5 RhD positive samples and 5 Del samples screened out by hospitals in Chengdu were sent to our laboratory for futher examination. The erythrocytes and buff coat were isolated, then DNA and RNA were extracted.All 10 samples were genotyped by PCR-SSP. After the mRNA was reversely transcribed into cDNA, the full-length mRNA of RHD and RHCE genes were simultaneously amplified by a pair of primers. Sanger sequencing and third-generation sequencing technology based on Nanopore were used to sequence the amplified products, and the types and expressions of different splices of RHD and RHCE gene mRNA transcripts were analyzed. Results The method established in this study can simultaneously amplify the full length transcripts of RHD and RHCE. Ten different RHD gene mRNA transcripts and nine RHCE gene mRNA transcripts were detected in 10 samples. RHD full-length transcript (RHD-201) can be detected in RhD Del type, but the expression amount was significantly lower than that in RhD positive samples. The expression amount of transcript RHD-207 (Del789) in Del samples was significantly higher than that in RhD positive samples. The transcript RHD-208 (Del8910+ 213) was only detected in RhD Del type individuals, and no significant difference was found between other RHD transcripts and all RHCE transcripts in the two phenotypes. Conclusion In this study, an analytical method for sequencing full-length transcript isomers of RHD and RHCE mRNA via the third generation was successfully established, and complex alternative splicing patterns were found in RHD and RHCE genes, providing a new method for the study of alternative splicing of blood group gene variants mRNA.