Systemic analysis and zygosity determination of the RHD gene in a D-negative Chinese Han population reveals a novel D-negative RHD gene

Background and Objectives The aim of this study was to systemically analyse the genetic background of D negativity in a Chinese Han population. Materials and Methods DNA of 74 D-negative samples was analysed by using an RHD multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by PCR-restriction fragment length polymorphism (PCR-RFLP) for RHD zygosity determination. Sixty-five samples were additionally analysed by using real-time quantitative PCR on RHD exon 7. RHD exon-specific sequencing was performed on discrepant samples. Results Forty-six samples (62%) showed the absence of RHD-specific exons by RHD MPX PCR and homozygous RHD negativity by PCR-RFLP. Twenty-two samples (30%) showed a 1227G>A mutation, characteristic for the Del phenotype. Five (70/6) samples showed all characteristics of the RHD(1-2)-CE(3-9)-D(10) hybrid gene. One sample (1(.)4%) showed a novel 933C>A nonsense mutation in RHD exon 6, which resulted in a premature stop codon. The RHD gene deletion, RHD-CE-D hybrid genes and one novel 93C>A mutation were found to be the three mechanisms that cause D negativity in our samples. The 1227G>A Del mutation was found to be the major cause of discrepant results between genotyping and phenotyping strategies, favouring genotyping of D-negative samples