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

The sedel allele is one of the nonsecretor alleles (se) of FUT2 generated by an Alu-mediated recombination event and was first found in Indian Bombay phenotype individuals who have anti-H, anti-A, and anti-B antibodies in their serum. As well as anti-A, and anti-B antibodies, anti-H is clinically significant because it causes sever hemolytic transfusion reactions. Like sedel, se302 having a missense single nucleotide polymorphism (SNP), 302C > T, is characteristic of South Asians with a frequency of 10–30%. We developed a real-time PCR melting curve analysis for detection of sedel using a 127-bp amplicon encompassing the breakpoint junction. In addition, by performing duplex PCR by amplifying a 65-bp amplicon of the FUT2 coding region at the same time, we could determine the zygosity of sedel in a single tube. We also developed an Eprobe-mediated PCR assay (Eprobe-PCR) for detection of 302C > T of FUT2. These methods were validated by analyzing 58 Tamils and 54 Sinhalese in Sri Lanka. Both the duplex PCR melting curve analysis for determination of sedel zygosity and the Eprobe-PCR assay for detection of 302C > T exactly determined three genotypes. In addition, the results of the present methods were in complete agreement with those obtained by previously established methods. The two present methods were reliable and seem to be advantageous for large-scale association studies of FUT2 polymorphisms in South Asian populations.

www.nature.com/scientificreports/ 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 , se 302 and se 428 were common se alleles as with other South Asian populations. The frequency of se del was reported to be 28 and 13%, that of se 302 was 9.5 and 27% and that of se 428 was 9.5 and 22% for Tamils and Sinhalese, respectively 12 .
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 B 12 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 instrument 19 .
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 (127bp) 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. www.nature.com/scientificreports/

Results
The Lewis phenotype on red cells of each individual (58 Tamil and 54 Sinhalese) had been determined previously 12 . Le(a − b +) was identified as a secretor and Le(a + b −) as a nonsecretor whereas discrimination of secretors from nonsecretors among 26 Le(a − b -) subjects by phenotyping is impossible. There was no discrepancy between phenotype and genotype determined by Sanger sequencing and denaturing high-performance liquid chromatography (dHPLC) in Lewis-positive subjects 12 . As described previously, we could define that 16 of 26 Le(a − b −) subjects were secretors and ten of them were nonsecretors by genotyping of the FUT2 12,20 .
PCR amplification of se del . First we amplified a PCR product using a set of primers encompassing a 127bp region of an se del breakpoint (sedel-F and sedel-R) on a real-time PCR platform (Fig. 1A). Specific amplification of the deletion breakpoint of se del was confirmed by an amplification signal only from individuals having se del and direct DNA sequencing of the PCR products of four selected subjects (data not shown).
Duplex real-time PCR method for detection of se del . We then designed a duplex real-time PCR to determine the zygosity of se del in a single tube. In addition to primers for the 127-bp se del -specific amplicon, we added primers for detection of FUT2 that lacked the se del allele in a single tube. We performed this on three selected individuals with genotypes of the wild type (+/+), heterozygote of se del (+/−), and homozygote of se del (−/−). Since these primers amplified a 65-bp region of the coding region of FUT2 (751-815 bp), the melting curve analysis of the duplex real-time PCR clearly distinguished the three genotypes from each other (data not shown). The melting temperature (Tm) value of the 127-bp amplicon of se del was around 85 °C, while that of 65-bp FUT2 coding region was around 81 °C. The lower limits of discrimination were around 16, 64, and 8 pg of DNA for −/− (homozygote of se del ), +/− (heterozygote of se del ), and +/+ (wild type), respectively (data not shown). We then applied this method to analyze 58 Tamil and 54 Sinhalese samples and clearly discriminated three genotypes (Fig. 2). However, the results of se del zygosity of the present method were different from those of the previous conventional PCR assay for one Tamil and two Sinhalese 12 . We reanalyzed these three individuals by conventional PCR. Although we had judged these three individuals to be the +/+ genotype by conventional PCR previously, reanalysis suggested that three individuals were the +/− genotype. Thus the results of se del zygosity determined by the present method were completely identical to those by the conventional PCR genotyping method, and the numbers of +/+, +/−, and −/− genotypes were 28, 27, and 3 for Tamils and 38, 16, and 0 for Sinhalese. The repeatability was confirmed by two independent assays.
Genotyping of 302C > T of FUT2 using Eprobe-PCR. Next, we performed melting curve genotyping using a 195-bp amplicon and a 25-bp Eprobe (see Fig. 1B). To amplify FUT2 specifically, we selected a reverse primer with a seven-base difference from SEC1 (which is identical to the reverse primer of an unlabeled probe based on high-resolution melt (HRM) analysis for detection of 385A > T of FUT2) 21 . Using this method, we clearly distinguished C/C (Tm: around 72 °C), C/T (Tm: around 63 °C and 72 °C), and T/T (Tm: around 63 °C) genotypes from each other (data not shown). We then applied it to 58 Tamils and 54 Sinhalese whose 302C > T genotypes had been determined by Sanger sequencing and dHPLC previously. As shown in Fig. 3, the three genotypes of 302C > T of FUT2 could be separated clearly, the results were fully in agreement with previous genotyping results, and the numbers of C/C, C/T, and T/T were 43 (including 19 individuals with C/− genotype), 7, and 6 (all 6 individuals were T/− genotype) in Tamils and 28 (including 13 individuals with C/− genotype), 19, and 7 (including 3 individuals with T/− genotype) in Sinhalese. The repeatability was confirmed by two independent assays. In addition, we did not obtain any amplification signal in real-time PCR of three Tamils with the −/− genotype. The results suggested the specific amplification of FUT2 but not SEC1.

Discussion
We recently developed several HRM-based real-time PCR methods for detection of se fus , se 428 , and se 385 alleles [21][22][23] . Predominant se alleles in several South Asian populations are se 428 , se 302 , and se del12 . In addition to these three se alleles, se 385 has relatively high frequency in Bangladeshi 11 . Therefore, we should genotype se 428 , se 302 , and se del in many South Asians, whereas se 428 , se 385 , se 302 , and se del should be genotyped in Bangladeshi for association studies of FUT2 8,24 . Even when we do not consider the deletion allele (se del ), the estimation of secretor status is not affected. For example, the Se/− genotype is judged as Se/Se and the se/− genotype as se/se. However, we need to be alert for se del when targeting a population with high frequency of it. In this study, we did not detect any real-time PCR amplification signal of 302C > T in three Tamils with the −/− genotype (Fig. 3A). The results persuaded us to screen for se del in South Asians; otherwise, we could not know whether the reason for the lack of an amplification signal of FUT2 was actually an absence of FUT2 or another problem, such as degradation of genomic DNA. In addition, it is likely that we overestimated homozygotes. In fact, without considering the results of se del screening, we misjudged the 302C/− genotype of 19 Tamils and 13 Sinhalese as C/C and 302T/− genotype of 6 Tamils and 3 Sinhalese as T/T by the Eprobe-PCR assay.
Unfortunately, we overlooked three individuals with the +/− genotype by previous conventional PCR for detection of an 1.8-kb amplicon because of the relatively large size of the PCR product and by simplex PCR without an amplification control 12 . On the other hand, the present duplex real-time PCR melting curve analysis included an amplification control (65-bp FUT2 coding sequence) that also allowed determination of se del zygosity in a single tube. Therefore, the present duplex real-time PCR assay for detection of se del is more reliable and faster than previous conventional PCR methods.
We previously developed a triplex TaqMan PCR assay to detect CNVs of FUT2 14 . The advantage of this method is not only detection of known CNVs but the potential to detect novel CNVs. However, it depends largely www.nature.com/scientificreports/ on both the quality and quantity of DNA to work well and carries a cost in terms of three probes. On the other hand, the real-time PCR melting curve analysis we present here is dedicated to detection of the se del and unable to detect other CNVs. The present assay is cost-effective, easy to use, straightforward, and not very dependent on both quality and quantity of DNA. Because a 164-bp sequence surrounding 302C > T is completely identical to that of SEC1 and this sequence contains another high frequency SNP (about 50% in global populations), 357C > T (rs281377, synonymous SNP), it was difficult to select appropriate primers for short amplicon HRM to detect 302C > T of FUT2. For this reason, in this study, we employed Eprobe-PCR instead of HRM analysis for detection of 302C > T of FUT2. Compared with HRM analysis using a short amplicon, Eprobe-PCR needs a labeled probe, and is therefore more expensive. However, the HRM method is based on detection of subtle differences of the melting curve and melting temperature of PCR amplicons, whereas the Eprobe-PCR method is based on detection of relatively large differences of the melting curve and melting temperature of a short probe sequence. Therefore, a probe-based melting curve analysis seems to be one of the most specific and sensitive methods to detect SNPs 25 . In fact, the Tm values of the wild-type (around 72 °C) and that of the mutant (around 63 °C) were quite different (around 9 °C). This significant difference made a clear distinction of the three genotypes of C/C, C/T, and T/T possible. Thus we believe that the present Eprobe-PCR for detection 302C > T of FUT2 is quite useful and reliable.
In conclusion, the present two protocols seem to be a reliable and high throughput method for detection of se del and se 302 in South Asian subjects and for examination of genetic basis of Indian Bombay phenotype in more detail. Duplex real-time PCR melting curve analysis for detection of se del . Since both the 5′ and 3′ deletion breakpoints of se del are located within Alu-repetitive elements 13 , the primers were carefully designed to amplify only a recombination allele (se del ) but not other Alu-elements using Primer 3 (https:// bioin fo. ut. ee/ prime r3-0. 4.0/) 26 and a BLAST search (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi) (Fig. 1A).    Fig. 1B underlined) and Eprobe (5′-TCT TCA GAA UCA CCC TGC CGG TGC T-3′-AmC3; U indicates the position of the modified T by thiazole orange, 284-308 bp of FUT2). The Eprobe was blocked on the 3′ end (3′-amino-modifier C3) to prevent extension during PCR. The primers were synthesized by Eurofins Genomics, and the Eprobe was synthesized by K.K. DNAFORM. We performed real-time PCR and melting curve analysis using a LightCycler 480 Instrument II. Asymmetric PCR amplification was performed in 10 μL reaction mixture including 1-10 ng of genomic DNA, 5 µL of E-Taq 2 × PCR Mix (K.K. DNAFORM), 50 nM of FUT2-302-F primer, 250 nM of FUT2-302-R primer, and 250 nM of the Eprobe. The thermal profile was as follows: one cycle at 95 °C for 30 s, followed by 50 cycles with denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s, and extension at 72 °C for 15 s. The fluorescence data for monitoring real-time PCR amplification were collected the end of the annealing step of each cyle using a filter (533-580 nm). The products were heated to 95 °C for 1 min, rapidly cooled to 45 °C for 1 min, and fluorescence data for melting curve analysis were collected over the range from 50 to 80 °C. The melting curve genotypes were automatically clustered into separate groups by LightCycler 480 Gene Scanning Software (Roche Diagnostics).