Introduction

The human FUT2 encodes the secretor type α(1,2)fucosyltransferase. This enzyme participates in synthesis of the H antigen, which is a precursor of A and B blood group antigens, on the surface of mucosa or in secretions. Secretor status is dominant over non-secretor status, and therefore the presence of at least one functional FUT2 allele (Se) results in the secretor phenotype, while the non-secretors are homozygotes of the null alleles (se) and lack ABH antigens in saliva and other body fluids. Weak-secretors are homozygotes of the weak-secretor allele (Sew, see Table 1) or heterozygotes of Sew/se and express lower amounts of these antigens than secretors do1,2.

Table 1 Distribution of SNPs and alleles of FUT2 in various populations.
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The prevalence of non- or weak secretors is about 20–25% in most geographic regions, and statistical analysis of genetic variations of this locus suggest that the relative frequencies of secretors and non-secretors (or weak secretors) have been maintained for a long time by balancing selection, and that various pathogens may be considered an important selective force3,4.

The coding sequence of FUT2 is not interrupted by an intron and locates solely in the second exon, which encodes a 343-amino acid protein. This makes it easy to determine the haplotypes of SNPs of the protein coding region5,6. Because we numbered the nucleotide positions of FUT2 using a short coding region (999 bp: 332 amino acids) in previous studies3,7, we also used this to describe SNPs or alleles for easy comparison, while we used a long coding region (1032 bp: 343 amino acids) that has an additional 33 bp at the 5′-region for in silico analyses only to predict the impact of SNPs on the protein.

Because the blood group antigens on the surface of mucosa or in secretions can be used as a scaffold for infection by some kinds of pathogens, secretor status has been reported to be associated with susceptibility to viruses such as norovirus and rotavirus8,9. In addition, genome-wide association studies revealed its association with Crohn’s disease, celiac disease, and inflammatory bowel disease10,11.

Many single nucleotide polymorphisms (SNPs) and recombinant null alleles have been identified so far4,12,13,14,15,16. Representative SNPs and their alleles are shown in Table 1. Interestingly, some of them are distributed in a population-specific pattern. For association studies on secretor status, we need to know the genetic background of FUT2 of the target populations unless we can determine the secretor status by phenotype. However, because amino acid substitutions do not always affect the activity of the encoded Se enzyme, predicting it is sometimes difficult17,18,19. To examine the impact of each of the SNPs on the enzyme activity, we can monitor the α(1,2)fucosyltransferase activity by transient expression in cultured cells, followed by measurement of the enzyme activity using 14C-labeled fucose or its acceptor or examination of the H antigen on the cell surface by flow cytometry17,20,21.

The 1000 Genome Project (https://www.internationalgenome.org/) is the largest public catalogue of human variation and genotype data22. Erythrogene v0.8 (27 Nov 2017) (http://www.erythrogene.com/) extracted the data of blood group alleles from 1000 Genomes and matched them against blood group reference lists23. For FUT2, 73 alleles are listed at present. To survey the uncharacterized nonfunctional alleles of FUT2, we selected 18 alleles listed in Erythrogene (80T>C; 357C>T; 385A>T, 357C>T; 403C>T; 950C>T,357C>T; 964A>G, 357C>T; 385A>T; 539G>A,357C>T; 610G>T, 40A>G; 357C>T; 616G>C , 357C>T; 715C>T, 357C>T; 856T>C; 863C>T, 370G>A, 58C>T; 357C>T, 357C>T; 566T>C, 418G>A; 863C>T, 357C>T; 980C>A, 357C>T; 385A>T; 980C>A, 357C>T; 544G>A; 771G>A, 205G>A; 357C>T, 357C>T; 542C>T, 262A>C; 357C>T) and obtained genomic DNA of 22 individuals who have these alleles with reference to Phase 3 of the 1000 Genomes Project (Table 2). In this study, we experimentally confirmed the nonsynonymous SNPs and their haplotypes (alleles). We also analyzed the encoded enzyme activities by transient expression assays. In addition, we predicted the effect of each nonsynonymous SNP on the protein by several in silico methods.

Table 2 DNA samples and relationship between FUT2 alleles on database and experimentally determined.
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Results

Sequence and haplotype determination of FUT2

To survey the nonfunctional alleles of FUT2 in the database, we determined the DNA sequence of the total coding region of the FUT2 gene of 22 individuals who were expected to have uncharacterized alleles according to Erythrogene23. We detected all of the registered SNPs without deficiency and excess in respective DNA samples in the database by direct Sanger sequencing of the FUT2 coding region and encountered 17 uncharacterized nonsynonymous SNPs that have not been identified yet: 58C>T, 80T>C, 205G>A, 262A>C, 370G>A, 539G>A, 542C>T, 544G>A, 566T>C, 610G>T, 616G>C, 715C>T, 856T>C, 863C>T, 964A>G, and 980C>A. In addition to these, the alleles of 357C>T, 403C>T, and 950C>T were also investigated, although 403C>T and 950C>T themselves have been identified in Latin Americans (Se357,403) and a Mongolian (se357,950), respectively17,19. We then determined their haplotypes by subcloning them into plasmids. However, sequencing the clones revealed that the haplotypes of ten of 18 alleles were different from those registered in the database, even when the distance between SNPs was only 154 bp (positions 385 and 539 in HG02130, positions 216 (of se428) and 370 in HG04017, Table 2). Among the ten alleles, three uncharacterized nonsynonymous SNPs (370G>A, 544G>A, and 863C>T) were found on the nonfunctional allele se428, and 403C>T and 950C>T were found on another chromosome (Table 2). Accordingly, we performed functional analyses of the 13 alleles shown in Table 3. Haplotyping revealed that 863C>T was on two different alleles, 357C>T; 856T>C; 863C>T (HG03973 (Indian Telugu in the UK)) and 428G>A (plus 4 additional SNPs); 863C>T (NA18908 (Yoruba in Ibadan)) (Table 2).

Table 3 Evaluation of candidates for nonfunctional FUT2 alleles by expression of cell surface H antigens or in silico analysis of the nonsynonymous SNP.
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Functional analyses of candidates of non-secretor alleles

To determine whether each uncharacterized FUT2 allele encodes a functional Se enzyme or not, we ligated each allele into the mammalian expression vector pcDNA3.1, then transfected it into COS-7 cells. The cell surface H antigen was examined by flow cytometry using a monoclonal antibody to H type 1–4 (1E3)24. We performed five independent transient expression experiments, and the representative result for each allele was shown in Fig. 1. The expression levels of H antigens on cell surface are shown in Table 3. The percentage of H antigen–positive cells transfected with pcDNA3.1 ligated with the FUT2 of 80T>C; 357C>T, 964A>G; 357C>T, 357C>T; 539G>A, 40A>G; 616G>C, 357C>T; 715C>T, 58C>T; 357C>T, 357C>T; 980C>A, 205G>A; 357C>T, and 357C>T; 542C>T were almost identical to those ligated with the positive control of Se357 (wild-type allele, 26.22 ± 5.06%). On the other hand, the percentage of pcDNA3.1 ligated with FUT2 of 566T>C (9.28 ± 0.99%) was less than half of the positive control but higher than that of se357,385 (weak-secretor allele, 4.86 ± 0.76%), while that of 262A>C; 357C>T (2.03 ± 0.43%) was lower than that of se357,385. In the same experimental condition, expression of the H antigen on cells transfected with pcDNA3.1-FUT2 of 610G>T or 357C>T; 856T>C; 863C>T was almost undetectable as was that of se428 (0.68 ± 0.19%, Table 3).

Figure 1
figure 1

Expression of H antigens in the COS-7 cells transfected with various FUT2 constructs. The FUT2 allele containing uncharacterized SNP(s) of the FUT2, Se357 (wild-type allele), se357,385 (weak-secretor allele), or se428 (negative control) subcloned into pcDNA3.1(+) plasmids was transfected into COS-7 cells. After 2 days of culture, the cells were incubated with 1E3 antibody, followed by incubation with FITC-conjugated goat anti-mouse IgM secondary antibody, and expression of H antigen on the cell surface was monitored by flow cytometry.

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Determination of causal SNP of FUT2-357C>T; 856T>C; 863C>T for inactivation of the encoded enzyme

As mentioned above, the expression study suggested that FUT2 of 357C>T; 856T>C; 863C>T was a nonfunctional allele. However, this allele contained two nonsynonymous SNPs, 856T>C and 863C>T, and it was unclear which SNP was involved in inactivation of the Se enzyme. We generated FUT2 of 357C>T; 856T>C and FUT2 of 357C>T; 863C>T by in vitro mutagenesis and performed transient expression and flow cytometry. As shown in Table 3, 856T>C completely impaired and 863C>T somewhat impaired the activity.

Estimation of significance of uncharacterized nonsynonymous SNPs

In addition to the transient expression study, we predicted the possible impacts of 14 amino acid substitutions on the functions of encoded proteins using four software programs.

The results of predictions were not always consistent with those of expression experiments (Table 3). When FUT2 of 610G>T, of 357C>T; 856T>C; 863C>T, and of 262A>C; 357C>T are considered to be nonfunctional alleles and the others functional, the ratio of estimated mismatch was 42.9%, 64.3%, 57.1%, and 64.3% by MutationTaster, MutationAssessor, PolyPhen-2, and SIFT, respectively. Of 14 SNPs, the predicted effects were matched for all software and experiments for M38T, R191Q, G215W, and Y297H, while they were completely mismatched for I333V, R250W, V200A, A80T, and P192L (Table 3). The software generally tended to overestimate the impacts of the nonsynonymous SNPs we tested here.

Discussion

Recently, public databases are available that provide genetic variation data, and websites dedicated to various purposes using these data are also accessible. The 1000 Genomes Project Phase 3 provides a list of variants and haplotypes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping23. One study reported that phasing and imputation for rare variants are unreliable, which likely reflects the limited sample size of the project data25. In this study, we observed discordance between the haplotypes on Erythrogene from the 1000 Genomes project and those experimentally phased haplotypes for ten out of 18 haplotypes. Actually, the FUT2 alleles we investigated have frequencies of 0.02 to 0.06% in the studied 2504 samples of 26 populations on 1000 Genomes, that is, one to three of 5008 alleles. That may be the reason for the discordance, but it should not be ignored in association studies on secretor status when its prevalence is at a certain level in the target populations. Previously, we identified 400G>A (rs370886251) of a functional FUT2 allele Se400 in a Samoan population with a frequency of 2.1% (one of 48 alleles)26. This allele was also observed in Indonesians at 1.2% (two of 166 alleles)27. In addition, we encountered it in six New Guinean populations with the relatively high prevalences of 10.6% (42 of 114 alleles) to 36.8% (7 of 66 alleles)28. These results suggest that 400G>A is distributed in Oceania and neighboring countries. However, 400G>A is not listed in 1000 Genomes and subsequently Erythrogene. These observations raise the possibility that some of the nonsynonymous SNPs we examined here may be present at unignorable frequencies in certain populations25.

The best way to evaluate the activity of the Se enzyme may be consideration in conjunction with phenotypic data using red blood cells or saliva as specimens. In the Lewis blood group, Le(a − b +) is identified as a secretor, Le(a + b −) as a non-secretor, and Le(a + b +) as a weak-secretor29. We can also measure the amount of ABO substances in saliva. However, we could not perform phenotyping and can only speculate on the secretor status based on the genetic information. In this study, we estimated the activity of the enzyme encoded by FUT2 alleles of interest by transient expression and subsequent flow cytometry for detection of cell surface H antigens expressed. In addition, the impacts of the target missense SNPs on the Se enzyme are also deduced by four software programs because we experienced a discrepancy between the results obtained by in vitro and those by in silico analyses17,18,19. In the results, we also observed discrepancies for six to nine out of 14 SNPs, while all of the SNPs reside in the catalytic domain. Previously, we identified a FUT2 allele with 357C>T; 685G>A in a Bangladeshi secretor individual with genotype of 357C>T; 685G>A/302C>T. The α(1,2)fucosyltransferase activity of the enzyme encoded by FUT2 of 357C>T; 685G>A was 8% of that of the wild-type allele26. From this observation, we categorized FUT2 of 566T>C as a secretor allele (Se566) and that of 262A>C; 357C>T as a weak secretor allele (se262,357, Sew), while the secretor status phenotypes were unknown and the 566T>C substitution seemed to partially impair enzyme activity (Table 3).

In this study, we used an academically isolated monoclonal antibody, but the H antigens could be evaluated by the commercially available UEA-1 lectin (Ulex europaeus agglutinin I)19,21,24. In addition, as mentioned earlier, the sequence of the FUT2 is coded solely in exon 2. This makes it easy to clone the coding sequence or determine the haplotype of the coding polymorphisms.

Considering all the factors mentioned above, experimental procedures such as the haplotyping by cloning and evaluation of activity of the Se enzyme are desirable for FUT2. However, the 1000 Genome Project provides a valuable catalogue of human genetic variation, and the Erythrogene focus on blood group systems also enables easy reference to the FUT2 alleles from the 1000 Genome Project. Actually, the existences of listed SNPs themselves were completely confirmed in the indicated samples. In addition, the software programs for prediction of the functional impacts of amino acid substitutions are valuable tools, especially for the proteins whose functions are hard to estimate experimentally. Accordingly, there is no doubting the helpfulness of these databases or software programs.

Conclusion

We identified two non-secretor alleles (se610and se357,856,863) and one weak secretor allele (se262,357) in samples in the 1000 Genome Project. Experimental phasing and expression studies are desirable for analysis of FUT2.

Materials and methods

The study protocol was approved by the ethical committees of Kurume University School of Medicine (No. 342).

DNA samples

Twenty-two genomic DNAs (HG00701, HG01121, HG01705, HG02130, HG02345, HG03279, HG03681, HG03973, HG04017 of the 1000 Genomes project, NA11920 of CEPH/Utah Pedigree 1423, NA18626, NA18908, NA18951, NA18974, NA19063, NA19067, NA19625, NA20520, NA20531, NA20532, NA20805, NA20891of International HapMap Project) were purchased from the Coriell Institute for Medical Research (Camden, NJ, USA) (Table 2).

PCR amplification of coding region of FUT2 and sequence analysis

The coding regions of FUT2 were amplified and directly sequenced as described previously17.

Haplotype determination of FUT2

To determine the haplotypes of individuals who were heterozygous at two or more positions, we cloned PCR products into a plasmid and sequenced the clones as described previously17.

Transient expression study and evaluation of impact of nonsynonymous SNPs of FUT2

To evaluate the significance of each of uncharacterized allele, a transient expression experiment was performed as described previously17. The FUT2 alleles containing each SNP concerned, 357C>T (Se357: wild-type allele), 171A>G and 216C>T and 428G>A and 739G>A and 960A>G (se428: nonsecretor allele) or 357C>T and 385A>T (se357,385: weak-secretor allele) were inserted into pcDNA3.1(+) vectors. Three μg of each construct together with 100 ng of the pGL3 Promoter was transfected into 5 × 105 COS-7 cells (African green monkey kidney fibroblast-like cell) by means of TransIT-X2 (Mirus Bio LLC, Madison, WI).After 2 days of culture, the cells were incubated with 1E3 antibody for H type 1–4, followed by incubation with FITC-conjugated goat anti-mouse IgM (Bethyl Laboratories, Montgomery, TX) secondary antibody, and expression of H antigen on the cell surface was monitored by flow cytometry (BD Accuri C6, Becton Dickinson, Franklin Lakes, NJ) as described previously18,24. The experiments were performed five times. About 1 × 105 cells were lysed and the firefly luciferase activity was assayed using ONE-Glo Luciferase Assay System (Promega, Madison, WI) and the similar transfection efficiency in each experiment was confirmed by the intensity of luciferase light (Table 3).

Prediction of impacts of nonsynonymous SNPs on Se enzyme

The effect of each nonsynonymous SNP of FUT2 on the function of the enzyme were predicted using four free software programs, MutationTaster (http://www.mutationtaster.org/)30, MutationAssessor (http://mutationassessor.org/r3/)31, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)32, and Sorting Intolerant From Tolerant (SIFT) (http://sift.jcvi.org/)33.

Introduction of reverse substitution in allele containing 357C>T; 856T>C; 863C>T

Because functional analysis showed one allele containing 357C>T; 856T>C; 863C>T to be a nonfunctional allele, site-specific mutagenesis of the plasmid DNA was performed using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs Japan Inc., Tokyo, Japan) to determine which substitution is responsible for inactivation of the enzyme. The primer set used for reverse substitution at 856 (C to T) was 5′-CTG GGC CGC ATA CCT CAT GGG-3′ (forward primer, 846–866 bp of FUT2, position 856 and 863 are underlined) and 5′-ATC CCG AAC GTC CCA ATG GTC ATG-3′ (reverse primer, 822–845 bp of FUT2). That for 863 (T to C) was 5′-GCA CAC CTC ACG GGC GGA GAC-3′ (forward primer, 853–873 bp, position 856 and 863 are underlined) and 5′-GGC CCA GAT CCC GAA CGT-3′ (reverse primer, 835–852 bp of FUT2). The plasmid pcDNA3.1(+) containing FUT2 of 357C>T; 856T>C; 863C>T was used as a template. All procedures were performed according to the manufacturer’s protocol. The cycling conditions were an initial denaturation at 98 °C for 30 s, followed by 25 cycles of denaturation at 98 °C for 10 s, annealing at 70 °C (for 856 reverse substitution) or 72 °C (for 863 reverse substitution) for 20 s, and extension at 72 °C for 3 min, followed by final extension at 72 °C for 2 min. The sequences of the isolated clones were confirmed. Finally, we obtained two constructs, pcDNA3.1(+) containing FUT2 of 357C>T; 856T>C and 357C>T; 863C>T.