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

被引：2

作者：

Wang, Yufeng ^{[1
,2
,3
]}

Chen, Xihui ^{[1
,2
]}

Chen, Qi ^{[1
,2
]}

Chen, Tangdong ^{[1
,2
]}

Chen, Kun ^{[4
]}

Wu, Yuanming ^{[1
,2
]}

Wang, Li ^{[1
,2
,5
]}

机构：

[1] Fourth Mil Med Univ, Dept Biochem & Mol Biol, Xian, Peoples R China

[2] Fourth Mil Med Univ, Shaanxi Prov Key Lab Clin Genet, Xian, Peoples R China

[3] Yanan Univ, Med Genet, Yanan, Peoples R China

[4] Fourth Mil Med Univ, KK Leung Brain Res Ctr, Dept Anat Histol & Embryol, Xian, Peoples R China

[5] Fourth Mil Med Univ, Sch Aerosp Med, Xian, Peoples R China

来源：

FRONTIERS IN GENETICS | 2022年 / 13卷

关键词：

HNA-3; TaqMan real-time polymerase chain reaction method; droplet digital PCR; genotype; EXPRESSION; VARIANTS;

D O I：

10.3389/fgene.2022.794285

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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.

引用

页数：8