rspcr什么时候用Rapid detection of phenotypes Bombay sedel and nonsecretor rs200157007 SNP (302C ＞ T) by real-time PCR-based methods

In humans, expression of the H antigen, a precursor of A and B antigens of the ABO blood group, is regulated by two α(1,2)fucosyltransferases. One is the H enzyme encoded by FUT1, which regulates expression of the H antigen, and thereafter ABO antigens, on the cell surface of erythrocytes and endothelial cells. The other is the Se enzyme, encoded by FUT2, which regulates expression of the H antigen on mucosal surfaces and in body fluids^1,2. Deficiency of the H enzyme (Bombay and para-Bombay phenotypes) is very rare, whereas deficiency of the Se enzyme (nonsecretor) is common¹. The Bombay phenotype individuals do not express H antigen and hence A antigen or B antigen either on red cells or in secretions, because they lack both H and Se enzyme activities. As a result, they have anti-H, anti-A, and anti-B antibodies in their serum and can receive only autologous blood or blood from another Bombay phenotype individual. Transfusing blood group O red cells to them can cause severe hemolytic transfusion reactions¹. Functional alleles of FUT1 (H) and FUT2 (Se) are dominant over nonfunctional alleles of FUT1 (h) and FUT2 (se)¹. FUT1 and FUT2 locate on chromosome 19q3.3 beside a pseudogene (SEC1) having high sequence similarity particularly to FUT2³.

Several single nucleotide polymorphisms (SNPs) of se alleles are distributed in a population-specific manner^4,5,6. The 428G > A nonsense SNP (rs601338, W143X) constituting se⁴²⁸ is common in Europeans, Africans, and West Asians with a frequency about 50% and in South Asians with a frequency of 10–30%. On the other hand, the 385A > T missense SNP (rs1047781, I129F) constituting a weak secretor allele (Se^w) is common in East and Southeast Asians with a frequency about 50%. In addition, the 302C > T missense SNP (rs200157007, I101P) constituting se³⁰² was first identified in a Thai population with a low frequency and was demonstrated to be exclusively encountered in South Asians with a frequency of 10–30%^5,7,8.

Five se alleles that resulted from copy number variations (CNVs) have also been identified. Four of them (se^del, se^del2, se^del3, se^del4) were complete deletions of the coding region, whereas one (se^fus) was generated by a homologous recombination between SEC1 and FUT2⁹. Among them, the se^del allele was first identified in subjects of the Indian Bombay phenotype¹⁰, and, like the se³⁰² allele, is known to be characteristic to South Asians with a frequency of 10–30%^11,12. This allele was generated by a homologous recombination between two Alu elements that are 10-kb away from each other (Fig. 1A). Alu elements are the most abundant repetitive elements, composing ~ 10% of the human genome. In order to detect se^del, we first designed a conventional PCR method to amplify a relatively long fragment (1.8-kb) and then developed a triplex hydrolysis probe (TaqMan) PCR assay to detect CNVs of FUT2^13,14. However, se^del, se^del2, se^del3, and se^del4 cannot be discriminated by the triplex TaqMan PCR assay.

Sri Lanka has a diverse ethnic composition and 74% are Sinhalese and 18% are Tamils. We have determined genetic diversity of the FUT2 previously for the same population in this study and found that se^del, se³⁰² and se⁴²⁸ were common se alleles as with other South Asian populations. The frequency of se^del was reported to be 28 and 13%, that of se³⁰² was 9.5 and 27% and that of se⁴²⁸ was 9.5 and 22% for Tamils and Sinhalese, respectively¹².

Recent studies suggested that FUT2 polymorphism (secretor status) is associated with susceptibility to various infectious diseases, such as norovirus, rotavirus, COVID-19, and several clinical conditions such as Crohn’s disease and low plasma vitamin B₁₂ levels^15,16,17,18. Large scale replication studies of various populations or independent samples are important for confirmation of these associations. Therefore, accurate and high-throughput genotyping should to be performed. However, common nonsecretor alleles are not shared by different continental populations.

An Eprobe-mediated PCR method (Eprobe-PCR) was recently developed for detection of SNPs. Eprobe is a hybridization-dependent fluorescence probe based on the quenching of two dye moieties in the condition of a single-stranded oligonucleotide and can be applied to sequential quantitative PCR, followed by melting curve analysis in a single reaction tube with a real-time PCR instrument¹⁹.

The aim of present study was to develop high-throughput methods for detection of FUT2 polymorphisms applicable to South Asians and to examine the molecular basis of Indian Bombay phenotype in more detail. For this purpose, we developed a duplex real-time PCR melting curve analysis for detection of se^del using a short (127-bp) amplicon together with the FUT2 coding region using a 65-bp amplicon to determine se^del zygosity in a single tube. We also developed an Eprobe-PCR method for detection of 302C > T of FUT2 using a 195-bp amplicon.