SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR

Abstract

Background: Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (CTL2, also known as SLC44A2). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare SLC44A2*2 allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of SLC44A2 in mixed populations with different proportions.Methods: DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in SLC44A2. The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. SLC44A2 frequency was determined by two-channel ddPCR.Results: The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of SLC44A2*1 and SLC44A2*2 in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%–70.8%) and 35.1% (95% CI, 29.2%–41%), respectively. The ratio of SLC44A2*2 alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the SLC44A2 frequency in the population to be accurately inferred.Conclusion: This new method of detecting SLC44A2 alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the SLC44A2 frequency by ddPCR using the probes that we designed.

Non-invasive prenatal testing for fetal Ss, Kidd, and CTL2 blood group prediction by multiplex digital droplet PCR

“… 28 The frequency of the Jk a blood group gene in the Chinese Han population reported in 2012 was 51%, which is close to the frequency of 48.47% found in our study. 29 The frequency of the HNA-3A blood group gene reported in 2022 in northwest China population was 64.90%, close to the frequency of 66.57% in our study. 30 Therefore, we propose that it is feasible to use ddPCR to calculate the frequency of blood group–related genes in the human population.…”

Application of droplet digital PCR combined with TaqMan real‐time PCR in Dombrock blood group genotyping in Northwest China

“…This study describes the application of droplet digital PCR combined with TaqMan real-time PCR for genotyping and gene frequency determination. This method is highly precise and accurate [27] for the inference of allele frequencies in the Dombrock blood group system, indicating that it can be extended to other blood groups with SNPs. In contrast to the conventional method of detecting the genotype of each donor, the use of blood pools can remarkably improve the efficiency of estimating gene frequency in large populations.…”