Rapid detection of sedel and 302C>T (rs200157007) responsible for nonsecretor phenotype by real-time PCR-based methods

Rapid detection of sedel and 302C>T (rs200157007) responsible for nonsecretor phenotype by real-time PCR-based methods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rapid detection of se del and 302C>T (rs200157007) responsible for nonsecretor phenotype by real-time PCR-based methods Mikiko Soejima, Yoshiro Koda This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-396764/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jul, 2021 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract The se del allele is one of the nonsecretor alleles ( se ) of FUT2 generated by an Alu-mediated recombination event and was first found in an Indian Bombay phenotype. Like se del , se 302 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 se del 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 se del 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 se del 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. Molecular Biology Molecular Genetics Medical Genetics FUT2 sedel rs200157007 nonsecretor allele South Asia Figures Figure 1 Figure 2 Figure 3 Introduction 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 common 1 . Functional alleles of FUT1 ( H ) and FUT2 ( Se ) are dominant over nonfunctional alleles of FUT1 ( h ) and FUT2 ( se ). FUT1 and FUT2 locate on chromosome 19q3.3 beside a pseudogene ( SEC1 ) having high sequence similarity particularly to FUT2 3 . Several single nucleotide polymorphisms (SNPs) of se alleles are distributed in a population-specific manner 4–6 . The 428G > A nonsense SNP (rs601338, W143X) constituting se 428 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 se 302 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 . 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 FUT2 8 . Among them, the se del allele was first identified in subjects of the Indian Bombay phenotype 9 , and, like the se 302 allele, is known to be characteristic to South Asians with a frequency of 10–30% 10,11 . This allele was generated by a homologous recombination between two Alu elements that are 10-kb away from each other (Fig. 1 A). 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 12,13 . However, se del , se del2 , se del3 , and se del4 cannot be discriminated by the triplex TaqMan PCR assay. 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 14 . The aim of present study was to develop and evaluate high-throughput methods for detection of FUT2 polymorphisms applicable to South Asians. 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. Results PCR amplification of se del First we amplified a PCR product using a set of primers encompassing a 127-bp region of an se del breakpoint (sedel-F and sedel-R) on a real-time PCR platform (Fig. 1 A, B). 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. 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 11 . 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. 1 B). 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 ) 15 . 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 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 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 16–19 . 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. For this purpose, we recently developed several HRM-based real-time PCR methods for detection of se fus , se 428 , and se 385 alleles 15,20,21 . Predominant se alleles in several South Asian populations are se 428 , se 302 , and se del 11 . In addition to these three se alleles, se 385 has relatively high frequency in Bangladeshi 10 . 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 22,23 . 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. 3 A). 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 11 . 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 13 . The advantage of this method is not only detection of known CNVs but the potential to detect novel CNVs. However, it depends largely 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 24 . 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. Materials And Methods Statements and DNA samples All methods were carried out in accordance with relevant guidelines and regulations. DNA samples from 58 Tamil and 54 Sinhalese in Sri Lanka whose FUT2 genotypes had been already determined were used 11 . The oral informed consent was obtained and the DNA samples were taken from participants in 2002. The statement for oral informed consent approved by ethical committee of Kurume University in 2002. However, present study protocol was approved by the ethical committee of Kurume University School of Medicine in 2017 using existing and already anonymized DNA samples (Bioethics approval No. 342). 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 12 , the primers were carefully designed to amplify only a recombination allele ( se del ) but not other Alu-elements using Primer 3 ( https://bioinfo.ut.ee/primer3-0.4.0/ 25 ) and a BLAST search ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) (Fig. 1 B). For amplification of the 127-bp amplicon of se del encompassing a homologous sequence of 25 bp at each breakpoint, we selected a sedel-F primer (5'-TCTCAGTAAGATAGACCAGGTGTGG-3', 35–11 bp upstream from the 5' end of the 25-bp homologous sequence) and a sedel-R primer (5'-GACAGAGTTTCACCATGTCAGC-3', 46–67 bp downstream from the 3' end of the 25-bp homologous sequence) from several candidates. For duplex PCR to determine the zygosity of se del , we added primers which were recently designed for HRM analysis (FUT2-778-F: 5'-TTTGCTGGCGATGGCATT-3' and FUT2-778-R: 5'-TGGTTACACTGTGTGAGTAGAGCAA-3') to detect 778C > del (P260Lfs*16, rs1799761) as FUT2 -specific primers 21 . These primers amplified the 751–815 bp coding region of FUT2 which lacked the se del allele (Fig. 1 A). The primers were synthesized by Eurofins Genomics (Tokyo, Japan). The 10 µl PCR reaction contained 1–10 ng genomic DNA, 5 µL of LightCycler 480 HRM Master mix (Roche Diagnostics, Mannheim, Germany), 2.5 mM MgCl 2 , 125 nM of sedel-F and sedel-R primers, and 187.5 nM of FUT2-778-F and FUT2-778-R primers. Touchdown PCR was performed on a LightCycler 480 instrument II (Roche Diagnostics). The thermal profile was as follows: one cycle at 95°C for 10 min, denaturation at 95°C for 10 sec, annealing at 64°C for 20 sec with touchdown every 0.5°C, and extension at 72°C for 20 sec for 12 cycles, and then 95°C for 10 sec, 58°C for 20 sec, and 72°C for 20 sec for 33 cycles. The fluorescence data for monitoring real-time PCR amplification were collected at the end of the annealing step of each cycle using a filter (465 nm–510 nm). The products were heated to 95°C for 1 min, rapidly cooled to 40°C for 1 min, and then the fluorescence data for melting curve analysis were collected over the range from 75 to 90°C, increasing at 0.02°C/sec with 25 acquisitions/sec. The melting curve genotypes were automatically clustered into separate groups by LightCycler 480 Gene Scanning Software (Roche Diagnostics). Sanger sequencing of PCR products of se del PCR amplification was performed using sedel-F and sedel-R primers and LightCycler 480 HRM Master mix as describe above. Direct sequencing of the PCR amplicons of se del was performed using each PCR primer as a sequence primer 4 . Eprobe-PCR for detection of 302C > T of FUT2 Nucleotide positions of the SEC1 and FUT2 genes are numbered as described previously 26 . For PCR amplification surrounding 302C > T, Primer3 ( https://bioinfo.ut.ee/primer3-0.4.0/ ) was also used to design primers for specific amplification of FUT2 . In addition, Edesign software ver.2.00 ( http://www.dnaform.com/edesign2/ ) provided by K.K. DNAFORM (Yokohama, Japan) was used to design an Eprobe for detection of 302C > T of FUT2 . For Eprobe-PCR, we used a FUT2-302-F primer (5'-CCCTGGCCAAGATGAACGG-3', 218–236 bp of FUT2 and identical with the corresponding SEC1 sequence, Fig. 1 C underlined) a FUT2-302-R primer (5'-CGGTGAAGCGGACGTACT-3', 395–412 bp of FUT2 , a 7-base difference from SEC1 , Fig. 1 C underlined) and Eprobe (5'-TCTTCAGAAUCACCCTGCCGGTGCT-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 sec, 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 sec. The fluorescence data for monitoring real-time PCR amplification were collected the end of the annealing step of each cyle using a filter (533 nm–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). Declarations Competing interests The authors declare no conflict of interest. Author contributions M.S. performed experiments and contributed to the data analysis and drafting of the manuscript. Y.K. planned the experiment, contributed to the data analysis and interpretation, and drafted the paper. Acknowledgements We thank Ms. Katherine Ono for the English editing of this manuscript. References 1 Daniels, G. in Human blood groups (ed Geoff Daniels) Ch. 2, 11-95 (Wiley-Blackwell, 2013). 2 Clausen, H. & Hakomori, S. ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution. Vox Sang 56 , 1-20, doi:10.1111/j.1423-0410.1989.tb03040.x (1989). 3 Rouquier, S. et al. Molecular cloning of a human genomic region containing the H blood group alpha(1,2)fucosyltransferase gene and two H locus-related DNA restriction fragments. Isolation of a candidate for the human Secretor blood group locus. J Biol Chem 270 , 4632-4639 (1995). 4 Soejima, M. & Koda, Y. Survey and characterization of nonfunctional alleles of FUT2 in a database. Sci Rep 11 , 3186, doi:10.1038/s41598-021-82895-w (2021). 5 Koda, Y. et al. Contrasting patterns of polymorphisms at the ABO-secretor gene (FUT2) and plasma alpha(1,3)fucosyltransferase gene (FUT6) in human populations. Genetics 158 , 747-756 (2001). 6 Ferrer-Admetlla, A. et al. A natural history of FUT2 polymorphism in humans. Mol Biol Evol 26 , 1993-2003, doi:10.1093/molbev/msp108 (2009). 7 Chang, J. G. et al. Molecular analysis of secretor type alpha(1,2)-fucosyltransferase gene mutations in the Chinese and Thai populations. Transfusion 39 , 1013-1017, doi:10.1046/j.1537-2995.1999.39091013.x (1999). 8 Soejima, M. & Koda, Y. Genetic variation of FUT2 in a Peruvian population: identification of a novel LTR-mediated deletion and characterization of 4 nonsynonymous single-nucleotide polymorphisms. Transfusion 59 , 2415-2421, doi:10.1111/trf.15298 (2019). 9 Fernandez-Mateos, P. et al. Point mutations and deletion responsible for the Bombay H null and the Reunion H weak blood groups. Vox Sang 75 , 37-46 (1998). 10 Pang, H. et al. Two distinct Alu-mediated deletions of the human ABO-secretor (FUT2) locus in Samoan and Bangladeshi populations. Hum Mutat 16 , 274, doi:10.1002/1098-1004(200009)16:33.0.CO;2-I (2000). 11 Soejima, M. & Koda, Y. Denaturing high-performance liquid chromatography-based genotyping and genetic variation of FUT2 in Sri Lanka. Transfusion 45 , 1934-1939, doi:10.1111/j.1537-2995.2005.00651.x (2005). 12 Koda, Y., Soejima, M., Johnson, P. H., Smart, E. & Kimura, H. An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype. Hum Genet 106 , 80-85 (2000). 13 Soejima, M. & Koda, Y. TaqMan-based real-time polymerase chain reaction for detection of FUT2 copy number variations: identification of novel Alu-mediated deletion. Transfusion 51 , 762-769, doi:10.1111/j.1537-2995.2010.02895.x (2011). 14 Hanami, T. et al. Eprobe mediated real-time PCR monitoring and melting curve analysis. PLoS One 8 , e70942, doi:10.1371/journal.pone.0070942 (2013). 15 Soejima, M. & Koda, Y. Detection of the weak-secretor rs1047781 (385A>T) single nucleotide polymorphism using an unlabeled probe high-resolution-melting-based method. Electrophoresis (in press) . 16 Loureiro Tonini, M. A. et al. FUT2, Secretor Status and FUT3 Polymorphisms of Children with Acute Diarrhea Infected with Rotavirus and Norovirus in Brazil. Viruses 12 , doi:10.3390/v12101084 (2020). 17 Valenti, L. et al. Association of ABO blood group and secretor phenotype with severe COVID-19. Transfusion 60 , 3067-3070, doi:10.1111/trf.16130 (2020). 18 McGovern, D. P. et al. Fucosyltransferase 2 (FUT2) non-secretor status is associated with Crohn's disease. Hum Mol Genet 19 , 3468-3476, doi:10.1093/hmg/ddq248 (2010). 19 Hazra, A. et al. Common variants of FUT2 are associated with plasma vitamin B12 levels. Nat Genet 40 , 1160-1162, doi:10.1038/ng.210 (2008). 20 Soejima, M. & Koda, Y. High-resolution melting analysis for detection of fusion allele of FUT2. Electrophoresis 42 , 315-318, doi:10.1002/elps.202000241 (2021). 21 Soejima, M. & Koda, Y. Estimation of secretor status of ABO antigens by high-resolution melting analysis of rs601338 (428G > A). Clin Chim Acta 517 , 86-91, doi:10.1016/j.cca.2021.02.019 (2021). 22 Parker, E. P. K. et al. FUT2 Secretor Status Is Not Associated With Oral Poliovirus Vaccine Immunogenicity in South Indian Infants. J Infect Dis 219 , 578-581, doi:10.1093/infdis/jiy553 (2019). 23 Mottram, L., Wiklund, G., Larson, G., Qadri, F. & Svennerholm, A. M. FUT2 non-secretor status is associated with altered susceptibility to symptomatic enterotoxigenic Escherichia coli infection in Bangladeshis. Sci Rep 7 , 10649, doi:10.1038/s41598-017-10854-5 (2017). 24 Erali, M., Voelkerding, K. V. & Wittwer, C. T. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85 , 50-58, doi:10.1016/j.yexmp.2008.03.012 (2008). 25 Untergasser, A. et al. Primer3--new capabilities and interfaces. Nucleic Acids Res 40 , e115, doi:10.1093/nar/gks596 (2012). 26 Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G. & Lowe, J. B. Sequence and expression of a candidate for the human Secretor blood group alpha(1,2)fucosyltransferase gene (FUT2). Homozygosity for an enzyme-inactivating nonsense mutation commonly correlates with the non-secretor phenotype. J Biol Chem 270 , 4640-4649 (1995). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 Jul, 2021 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 15 Jun, 2021 Reviews received at journal 06 Jun, 2021 Reviewers agreed at journal 27 May, 2021 Reviewers agreed at journal 26 May, 2021 Reviewers invited by journal 26 May, 2021 Editor assigned by journal 24 May, 2021 Editor invited by journal 09 Apr, 2021 Submission checks completed at journal 09 Apr, 2021 First submitted to journal 06 Apr, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-396764","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":20541821,"identity":"82cf7f8c-3320-4c86-aa6c-ade6e1d4589b","order_by":0,"name":"Mikiko Soejima","email":"","orcid":"","institution":"Kurume University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mikiko","middleName":"","lastName":"Soejima","suffix":""},{"id":20541822,"identity":"564d054b-9f29-4d6b-b51e-85aa5610e55b","order_by":1,"name":"Yoshiro Koda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACHgYGCYYKIOMAG4o4G3blcC1nSNbC2IapBTcwOHP44I2f87bJ8R1gS5OubNsmxyCRwPjhBwNfHk4tZ9uSLXu33TaWPMB2TPJs221joBZmyR4GtmKcWs7zmEnwbruduOEAe5tkY9vtxP03EhikgX5JbMCjRfLvnNv1MC31DUBbfuPVcrbHTJq34XaCAchhQC0JQIex4bVF8syxZGuZY7cNZx5mS7ZsOHfbsIHnYZtljwFuv/CdST54803NbXm+422GNxvKbsszsCcfvvGj4hjOEFM4AGMxw8UYgU4yOJaAS4s8LhfX4NQyCkbBKBgFIw4AAESPWGb4KcIYAAAAAElFTkSuQmCC","orcid":"","institution":"Kurume University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yoshiro","middleName":"","lastName":"Koda","suffix":""}],"badges":[],"createdAt":"2021-04-06 06:44:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-396764/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-396764/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-021-94659-7","type":"published","date":"2021-07-22T15:01:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":7921668,"identity":"62471fd4-c727-4fb3-b72d-92032334a4c2","added_by":"auto","created_at":"2021-04-12 17:37:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115087,"visible":true,"origin":"","legend":"(A) Relative positions of the deleted regions of sedel. Two exons are indicated by white boxes, and the protein coding region is indicated by a black box. Positions of PCR primers for duplex real-time PCR are shown by black arrows. (B) Alignment of DNA sequences of amplified regions of 127-bp encompassing the sedel breakpoint. DNA sequences of the 5' breakpoint (5' sequence), 3' breakpoint (3' sequence), and junction region (Junction) within Alu elements are indicated. Gray boxes indicate 25-bp homologous sequence breakpoints at the 5' and 3' ends of the deletion. Primer sequences are underlined. An asterisk shows an identical nucleotide among the three sequences. (C) Alignment of DNA sequences of FUT2 (FUT2-C: allele of C, FUT2-T: allele of T at 302C\u003eT (rs200157007)), and corresponding regions of SEC1 are indicated. The Eprobe sequence is boxed. Primer sequences are underlined. An asterisk shows an identical nucleotide among three sequences.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-396764/v1/663c96c248071341025ca1d2.jpg"},{"id":7921462,"identity":"72f6346d-c86c-4f0f-8f12-90561769c1c5","added_by":"auto","created_at":"2021-04-12 17:34:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73094,"visible":true,"origin":"","legend":"Melting peak profiles of duplex PCR for detection of sedel zygosity. Melting peak profiles obtained for 58 randomly selected Tamils (A) and 54 \nrandomly selected Sinhalese (B). The individuals with genotypes of +/+ (wild type, blue), +/- (heterozygote of sedel, red), and -/- (homozygote of sedel, green) were completely separated by melt curve genotyping.\n","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-396764/v1/cdc4483d84a766947529451d.jpg"},{"id":7921669,"identity":"4f87d043-7b9f-4b30-9f9b-c6950ed3baab","added_by":"auto","created_at":"2021-04-12 17:37:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79107,"visible":true,"origin":"","legend":"Melting peak profiles of Eprobe-PCR for detection of 302C\u003eT of FUT2. Melting peak profiles obtained for 58 Tamils (A) and 54 Sinhalese (B). The individuals with genotypes of C/C (blue), C/T(red), and T/T (green) at rs200157007 were completely separated by melt curve genotyping. In addition, no amplification signal (and therefore no melting peak) was obtained in three Tamils who were homozygous for sedel (light blue).","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-396764/v1/e83485915924be45ae7394e2.jpg"},{"id":15672295,"identity":"b71863c3-ee4c-42ca-83d3-9528097f14dd","added_by":"auto","created_at":"2021-11-18 14:11:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":386393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-396764/v1/da399d5d-78d6-47c3-8fe2-d943174ad787.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eRapid detection of se\u003csup\u003edel\u003c/sup\u003e and 302C\u0026gt;T (rs200157007) responsible for nonsecretor phenotype by real-time PCR-based methods\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn humans, expression of the H antigen, a precursor of A and B antigens of the ABO blood group, is regulated by two \u0026alpha;(1,2)fucosyltransferases. One is the H enzyme encoded by \u003cem\u003eFUT1\u003c/em\u003e, 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 \u003cem\u003eFUT2\u003c/em\u003e, which regulates expression of the H antigen on mucosal surfaces and in body fluids\u003csup\u003e1,2\u003c/sup\u003e. Deficiency of the H enzyme (Bombay and para-Bombay phenotypes) is very rare, whereas deficiency of the Se enzyme (nonsecretor) is common\u003csup\u003e1\u003c/sup\u003e. Functional alleles of \u003cem\u003eFUT1\u003c/em\u003e (\u003cem\u003eH\u003c/em\u003e) and \u003cem\u003eFUT2\u003c/em\u003e (\u003cem\u003eSe\u003c/em\u003e) are dominant over nonfunctional alleles of \u003cem\u003eFUT1\u003c/em\u003e (\u003cem\u003eh\u003c/em\u003e) and \u003cem\u003eFUT2\u003c/em\u003e (\u003cem\u003ese\u003c/em\u003e). \u003cem\u003eFUT1\u003c/em\u003e and \u003cem\u003eFUT2\u003c/em\u003e locate on chromosome 19q3.3 beside a pseudogene (\u003cem\u003eSEC1\u003c/em\u003e) having high sequence similarity particularly to \u003cem\u003eFUT2\u003c/em\u003e\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSeveral single nucleotide polymorphisms (SNPs) of \u003cem\u003ese\u003c/em\u003e alleles are distributed in a population-specific manner\u003csup\u003e4\u0026ndash;6\u003c/sup\u003e. The 428G\u0026thinsp;\u0026gt;\u0026thinsp;A nonsense SNP (rs601338, W143X) constituting \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e428\u003c/em\u003e\u003c/sup\u003e is common in Europeans, Africans, and West Asians with a frequency about 50% and in South Asians with a frequency of 10\u0026ndash;30%. On the other hand, the 385A\u0026thinsp;\u0026gt;\u0026thinsp;T missense SNP (rs1047781, I129F) constituting a weak secretor allele (\u003cem\u003eSe\u003c/em\u003e\u003csup\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sup\u003e) is common in East and Southeast Asians with a frequency about 50%. In addition, the 302C\u0026thinsp;\u0026gt;\u0026thinsp;T missense SNP (rs200157007, I101P) constituting \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e302\u003c/em\u003e\u003c/sup\u003e 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\u0026ndash;30%\u003csup\u003e5,7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFive \u003cem\u003ese\u003c/em\u003e alleles that resulted from copy number variations (CNVs) have also been identified. Four of them (\u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel2\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel3\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel4\u003c/em\u003e\u003c/sup\u003e) were complete deletions of the coding region, whereas one (\u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003efus\u003c/em\u003e\u003c/sup\u003e) was generated by a homologous recombination between \u003cem\u003eSEC1\u003c/em\u003e and \u003cem\u003eFUT2\u003c/em\u003e\u003csup\u003e8\u003c/sup\u003e. Among them, the \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e allele was first identified in subjects of the Indian Bombay phenotype\u003csup\u003e9\u003c/sup\u003e, and, like the \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e302\u003c/em\u003e\u003c/sup\u003e allele, is known to be characteristic to South Asians with a frequency of 10\u0026ndash;30%\u003csup\u003e10,11\u003c/sup\u003e. This allele was generated by a homologous recombination between two Alu elements that are 10-kb away from each other (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Alu elements are the most abundant repetitive elements, composing\u0026thinsp;~\u0026thinsp;10% of the human genome. In order to detect \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e, 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 \u003cem\u003eFUT2\u003c/em\u003e\u003csup\u003e12,13\u003c/sup\u003e. However, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel2\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel3\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel4\u003c/em\u003e\u003c/sup\u003e cannot be discriminated by the triplex TaqMan PCR assay.\u003c/p\u003e\n\u003cp\u003eAn 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\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe aim of present study was to develop and evaluate high-throughput methods for detection of \u003cem\u003eFUT2\u003c/em\u003e polymorphisms applicable to South Asians. For this purpose, we developed a duplex real-time PCR melting curve analysis for detection of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e using a short (127-bp) amplicon together with the \u003cem\u003eFUT2\u003c/em\u003e coding region using a 65-bp amplicon to determine \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e zygosity in a single tube. We also developed an Eprobe-PCR method for detection of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2\u003c/em\u003e using a 195-bp amplicon.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003ePCR amplification of\u003c/em\u003e se\u003csup\u003edel\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eFirst we amplified a PCR product using a set of primers encompassing a 127-bp region of an \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e breakpoint (sedel-F and sedel-R) on a real-time PCR platform (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Specific amplification of the deletion breakpoint of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e was confirmed by an amplification signal only from individuals having \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e and direct DNA sequencing of the PCR products of four selected subjects (data not shown).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDuplex real-time PCR method for detection of\u003c/em\u003e se\u003csup\u003edel\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eWe then designed a duplex real-time PCR to determine the zygosity of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e in a single tube. In addition to primers for the 127-bp \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e-specific amplicon, we added primers for detection of \u003cem\u003eFUT2\u003c/em\u003e that lacked the \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e allele in a single tube. We performed this on three selected individuals with genotypes of the wild type (+/+), heterozygote of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e (+/-), and homozygote of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e (-/-). Since these primers amplified a 65-bp region of the coding region of \u003cem\u003eFUT2\u003c/em\u003e (751\u0026ndash;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 \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e was around 85\u0026deg;C, while that of 65-bp \u003cem\u003eFUT2\u003c/em\u003e coding region was around 81\u0026deg;C. We then applied this method to analyze 58 Tamil and 54 Sinhalese samples and clearly discriminated three genotypes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). However, the results of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e zygosity of the present method were different from those of the previous conventional PCR assay for one Tamil and two Sinhalese\u003csup\u003e11\u003c/sup\u003e. 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 \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenotyping of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of\u003c/em\u003e FUT2 \u003cem\u003eusing Eprobe-PCR\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNext, we performed melting curve genotyping using a 195-bp amplicon and a 25-bp Eprobe (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). To amplify \u003cem\u003eFUT2\u003c/em\u003e specifically, we selected a reverse primer with a seven-base difference from \u003cem\u003eSEC1\u003c/em\u003e (which is identical to the reverse primer of an unlabeled probe based on high-resolution melt (HRM) analysis for detection of 385A\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2\u003c/em\u003e) \u003csup\u003e15\u003c/sup\u003e. Using this method, we clearly distinguished C/C (Tm: around 72\u0026deg;C), C/T (Tm: around 63\u0026deg;C and 72\u0026deg;C), and T/T (Tm: around 63\u0026deg;C) genotypes from each other (data not shown). We then applied it to 58 Tamils and 54 Sinhalese whose 302C\u0026thinsp;\u0026gt;\u0026thinsp;T genotypes had been determined previously. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the three genotypes of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2\u003c/em\u003e 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 \u003cem\u003eFUT2\u003c/em\u003e but not \u003cem\u003eSEC1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecent studies suggested that \u003cem\u003eFUT2\u003c/em\u003e 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\u003csub\u003e12\u003c/sub\u003e levels\u003csup\u003e16\u0026ndash;19\u003c/sup\u003e. 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.\u003c/p\u003e\n\u003cp\u003eFor this purpose, we recently developed several HRM-based real-time PCR methods for detection of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003efus\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e428\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e385\u003c/em\u003e\u003c/sup\u003e alleles\u003csup\u003e15,20,21\u003c/sup\u003e. Predominant \u003cem\u003ese\u003c/em\u003e alleles in several South Asian populations are \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e428\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e302\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e11\u003c/sup\u003e. In addition to these three \u003cem\u003ese\u003c/em\u003e alleles, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e385\u003c/em\u003e\u003c/sup\u003e has relatively high frequency in Bangladeshi\u003csup\u003e10\u003c/sup\u003e. Therefore, we should genotype \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e428\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e302\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e in many South Asians, whereas \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e428\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e385\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e302\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e should be genotyped in Bangladeshi for association studies of \u003cem\u003eFUT2\u003c/em\u003e\u003csup\u003e22,23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEven when we do not consider the deletion allele (\u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e), the estimation of secretor status is not affected. For example, the \u003cem\u003eSe\u003c/em\u003e/- genotype is judged as \u003cem\u003eSe\u003c/em\u003e/\u003cem\u003eSe\u003c/em\u003e and the \u003cem\u003ese\u003c/em\u003e/- genotype as \u003cem\u003ese\u003c/em\u003e/\u003cem\u003ese\u003c/em\u003e. However, we need to be alert for \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e when targeting a population with high frequency of it. In this study, we did not detect any real-time PCR amplification signal of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T in three Tamils with the -/- genotype (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results persuaded us to screen for \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e in South Asians; otherwise, we could not know whether the reason for the lack of an amplification signal of \u003cem\u003eFUT2\u003c/em\u003e was actually an absence of \u003cem\u003eFUT2\u003c/em\u003e 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 \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e screening, we misjudged the 302C/\u003cem\u003e-\u003c/em\u003e genotype of 19 Tamils and 13 Sinhalese as C/C and 302T/\u003cem\u003e-\u003c/em\u003e genotype of 6 Tamils and 3 Sinhalese as T/T by the Eprobe-PCR assay.\u003c/p\u003e\n\u003cp\u003eUnfortunately, 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\u003csup\u003e11\u003c/sup\u003e. On the other hand, the present duplex real-time PCR melting curve analysis included an amplification control (65-bp \u003cem\u003eFUT2\u003c/em\u003e coding sequence) that also allowed determination of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e zygosity in a single tube. Therefore, the present duplex real-time PCR assay for detection of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e is more reliable and faster than previous conventional PCR methods.\u003c/p\u003e\n\u003cp\u003eWe previously developed a triplex TaqMan PCR assay to detect CNVs of \u003cem\u003eFUT2\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e. The advantage of this method is not only detection of known CNVs but the potential to detect novel CNVs. However, it depends largely 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 \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003eBecause a 164-bp sequence surrounding 302C\u0026thinsp;\u0026gt;\u0026thinsp;T is completely identical to that of \u003cem\u003eSEC1\u003c/em\u003e and this sequence contains another high frequency SNP (about 50% in global populations), 357C\u0026thinsp;\u0026gt;\u0026thinsp;T (rs281377, synonymous SNP), it was difficult to select appropriate primers for short amplicon HRM to detect 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2\u003c/em\u003e. For this reason, in this study, we employed Eprobe-PCR instead of HRM analysis for detection of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2\u003c/em\u003e. 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\u003csup\u003e24\u003c/sup\u003e. In fact, the Tm values of the wild-type (around 72\u0026deg;C) and that of the mutant (around 63\u0026deg;C) were quite different (around 9\u0026deg;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\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2\u003c/em\u003e is quite useful and reliable.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the present two protocols seem to be a reliable and high throughput method for detection of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e302\u003c/em\u003e\u003c/sup\u003e in South Asian subjects.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cem\u003eStatements and DNA samples\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll methods were carried out in accordance with relevant guidelines and regulations. DNA samples from 58 Tamil and 54 Sinhalese in Sri Lanka whose \u003cem\u003eFUT2\u003c/em\u003e genotypes had been already determined were used\u003csup\u003e11\u003c/sup\u003e. The oral informed consent was obtained and the DNA samples were taken from participants in 2002. The statement for oral informed consent approved by ethical committee of Kurume University in 2002. However, present study protocol was approved by the ethical committee of Kurume University School of Medicine in 2017 using existing and already anonymized DNA samples (Bioethics approval No. 342).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDuplex real-time PCR melting curve analysis for detection of\u003c/em\u003e se\u003csup\u003edel\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSince both the 5' and 3' deletion breakpoints of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e are located within Alu-repetitive elements\u003csup\u003e12\u003c/sup\u003e, the primers were carefully designed to amplify only a recombination allele (\u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e) but not other Alu-elements using Primer 3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfo.ut.ee/primer3-0.4.0/\u003c/span\u003e\u003c/span\u003e\u003csup\u003e25\u003c/sup\u003e) and a BLAST search (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). For amplification of the 127-bp amplicon of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e encompassing a homologous sequence of 25 bp at each breakpoint, we selected a sedel-F primer (5'-TCTCAGTAAGATAGACCAGGTGTGG-3', 35\u0026ndash;11 bp upstream from the 5' end of the 25-bp homologous sequence) and a sedel-R primer (5'-GACAGAGTTTCACCATGTCAGC-3', 46\u0026ndash;67 bp downstream from the 3' end of the 25-bp homologous sequence) from several candidates. For duplex PCR to determine the zygosity of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e, we added primers which were recently designed for HRM analysis (FUT2-778-F: 5'-TTTGCTGGCGATGGCATT-3' and FUT2-778-R: 5'-TGGTTACACTGTGTGAGTAGAGCAA-3') to detect 778C\u0026thinsp;\u0026gt;\u0026thinsp;del (P260Lfs*16, rs1799761) as \u003cem\u003eFUT2\u003c/em\u003e-specific primers\u003csup\u003e21\u003c/sup\u003e. These primers amplified the 751\u0026ndash;815 bp coding region of \u003cem\u003eFUT2\u003c/em\u003e which lacked the \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The primers were synthesized by Eurofins Genomics (Tokyo, Japan). The 10 \u0026micro;l PCR reaction contained 1\u0026ndash;10 ng genomic DNA, 5 \u0026micro;L of LightCycler 480 HRM Master mix (Roche Diagnostics, Mannheim, Germany), 2.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 125 nM of sedel-F and sedel-R primers, and 187.5 nM of FUT2-778-F and FUT2-778-R primers. Touchdown PCR was performed on a LightCycler 480 instrument II (Roche Diagnostics). The thermal profile was as follows: one cycle at 95\u0026deg;C for 10 min, denaturation at 95\u0026deg;C for 10 sec, annealing at 64\u0026deg;C for 20 sec with touchdown every 0.5\u0026deg;C, and extension at 72\u0026deg;C for 20 sec for 12 cycles, and then 95\u0026deg;C for 10 sec, 58\u0026deg;C for 20 sec, and 72\u0026deg;C for 20 sec for 33 cycles. The fluorescence data for monitoring real-time PCR amplification were collected at the end of the annealing step of each cycle using a filter (465 nm\u0026ndash;510 nm). The products were heated to 95\u0026deg;C for 1 min, rapidly cooled to 40\u0026deg;C for 1 min, and then the fluorescence data for melting curve analysis were collected over the range from 75 to 90\u0026deg;C, increasing at 0.02\u0026deg;C/sec with 25 acquisitions/sec. The melting curve genotypes were automatically clustered into separate groups by LightCycler 480 Gene Scanning Software (Roche Diagnostics).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSanger sequencing of PCR products of\u003c/em\u003e se\u003csup\u003edel\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ePCR amplification was performed using sedel-F and sedel-R primers and LightCycler 480 HRM Master mix as describe above. Direct sequencing of the PCR amplicons of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e was performed using each PCR primer as a sequence primer \u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEprobe-PCR for detection of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of\u003c/em\u003e FUT2\u003c/p\u003e\n\u003cp\u003eNucleotide positions of the \u003cem\u003eSEC1\u003c/em\u003e and \u003cem\u003eFUT2\u003c/em\u003e genes are numbered as described previously\u003csup\u003e26\u003c/sup\u003e. For PCR amplification surrounding 302C\u0026thinsp;\u0026gt;\u0026thinsp;T, Primer3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfo.ut.ee/primer3-0.4.0/\u003c/span\u003e\u003c/span\u003e) was also used to design primers for specific amplification of \u003cem\u003eFUT2\u003c/em\u003e. In addition, Edesign software ver.2.00 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.dnaform.com/edesign2/\u003c/span\u003e\u003c/span\u003e) provided by K.K. DNAFORM (Yokohama, Japan) was used to design an Eprobe for detection of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2\u003c/em\u003e. For Eprobe-PCR, we used a FUT2-302-F primer (5'-CCCTGGCCAAGATGAACGG-3', 218\u0026ndash;236 bp of \u003cem\u003eFUT2\u003c/em\u003e and identical with the corresponding \u003cem\u003eSEC1\u003c/em\u003e sequence, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC underlined) a FUT2-302-R primer (5'-CGGTGAAGCGGACGTACT-3', 395\u0026ndash;412 bp of \u003cem\u003eFUT2\u003c/em\u003e, a 7-base difference from \u003cem\u003eSEC1\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC underlined) and Eprobe (5'-TCTTCAGAAUCACCCTGCCGGTGCT-3'-AmC3; U indicates the position of the modified T by thiazole orange, 284\u0026ndash;308 bp of \u003cem\u003eFUT2\u003c/em\u003e). 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 \u0026micro;L reaction mixture including 1\u0026ndash;10 ng of genomic DNA, 5 \u0026micro;L of E-Taq 2\u0026times;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\u0026deg;C for 30 sec, followed by 50 cycles with denaturation at 95\u0026deg;C for 15 s, annealing at 58\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 15 sec. The fluorescence data for monitoring real-time PCR amplification were collected the end of the annealing step of each cyle using a filter (533 nm\u0026ndash;580 nm). The products were heated to 95\u0026deg;C for 1 min, rapidly cooled to 45\u0026deg;C for 1 min, and fluorescence data for melting curve analysis were collected over the range from 50 to 80\u0026deg;C. The melting curve genotypes were automatically clustered into separate groups by LightCycler 480 Gene Scanning Software (Roche Diagnostics).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.S. performed experiments and contributed to the data analysis and drafting of the manuscript. Y.K. planned the experiment, contributed to the data analysis and interpretation, and drafted the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ms. Katherine Ono for the English editing of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Daniels, G. in \u003cem\u003eHuman blood groups\u003c/em\u003e\u0026nbsp;\u0026nbsp; (ed Geoff Daniels) Ch. 2, 11-95 (Wiley-Blackwell, 2013).\u003c/p\u003e\n\u003cp\u003e2\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Clausen, H. \u0026amp; Hakomori, S. 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K.\u003cem\u003e et al.\u003c/em\u003e FUT2 Secretor Status Is Not Associated With Oral Poliovirus Vaccine Immunogenicity in South Indian Infants. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e219\u003c/strong\u003e, 578-581, doi:10.1093/infdis/jiy553 (2019).\u003c/p\u003e\n\u003cp\u003e23\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mottram, L., Wiklund, G., Larson, G., Qadri, F. \u0026amp; Svennerholm, A. M. FUT2 non-secretor status is associated with altered susceptibility to symptomatic enterotoxigenic Escherichia coli infection in Bangladeshis. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 10649, doi:10.1038/s41598-017-10854-5 (2017).\u003c/p\u003e\n\u003cp\u003e24\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Erali, M., Voelkerding, K. V. \u0026amp; Wittwer, C. T. High resolution melting applications for clinical laboratory medicine. \u003cem\u003eExp Mol Pathol\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 50-58, doi:10.1016/j.yexmp.2008.03.012 (2008).\u003c/p\u003e\n\u003cp\u003e25\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Untergasser, A.\u003cem\u003e et al.\u003c/em\u003e Primer3--new capabilities and interfaces. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, e115, doi:10.1093/nar/gks596 (2012).\u003c/p\u003e\n\u003cp\u003e26\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G. \u0026amp; Lowe, J. B. Sequence and expression of a candidate for the human Secretor blood group alpha(1,2)fucosyltransferase gene (FUT2). Homozygosity for an enzyme-inactivating nonsense mutation commonly correlates with the non-secretor phenotype. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e270\u003c/strong\u003e, 4640-4649 (1995).\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"FUT2, sedel, rs200157007, nonsecretor allele, South Asia","lastPublishedDoi":"10.21203/rs.3.rs-396764/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-396764/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e allele is one of the nonsecretor alleles (\u003cem\u003ese\u003c/em\u003e) of \u003cem\u003eFUT2\u003c/em\u003e generated by an Alu-mediated recombination event and was first found in an Indian Bombay phenotype. Like \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003e302\u003c/em\u003e\u003c/sup\u003e having a missense single nucleotide polymorphism (SNP), 302C\u0026thinsp;\u0026gt;\u0026thinsp;T, is characteristic of South Asians with a frequency of 10\u0026ndash;30%. We developed a real-time PCR melting curve analysis for detection of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e using a 127-bp amplicon encompassing the breakpoint junction. In addition, by performing duplex PCR by amplifying a 65-bp amplicon of the \u003cem\u003eFUT2\u003c/em\u003e coding region at the same time, we could determine the zygosity of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e in a single tube. We also developed an Eprobe-mediated PCR assay (Eprobe-PCR) for detection of 302C\u0026thinsp;\u0026gt;\u0026thinsp;T of \u003cem\u003eFUT2.\u003c/em\u003e These methods were validated by analyzing 58 Tamils and 54 Sinhalese in Sri Lanka. Both the duplex PCR melting curve analysis for determination of \u003cem\u003ese\u003c/em\u003e\u003csup\u003e\u003cem\u003edel\u003c/em\u003e\u003c/sup\u003e zygosity and the Eprobe-PCR assay for detection of 302C\u0026thinsp;\u0026gt;\u0026thinsp;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 \u003cem\u003eFUT2\u003c/em\u003e polymorphisms in South Asian populations.\u003c/p\u003e","manuscriptTitle":"Rapid detection of sedel and 302C\u0026gt;T (rs200157007) responsible for nonsecretor phenotype by real-time PCR-based methods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-12 17:34:57","doi":"10.21203/rs.3.rs-396764/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-06-15T18:15:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-06-06T23:46:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"041b32cb-b59f-420f-8aed-bc06a691e009","date":"2021-05-27T13:43:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"062a1e1b-9758-4d75-b243-bd203ced9775","date":"2021-05-27T01:01:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-05-26T16:08:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-05-24T10:10:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-04-09T21:58:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-04-09T10:09:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-04-06T06:33:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc15a157-810e-4f0e-8b28-222f9e53fcc2","owner":[],"postedDate":"April 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":3586390,"name":"Molecular Biology"},{"id":3586391,"name":"Molecular Genetics"},{"id":3586392,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2021-08-22T15:11:53+00:00","versionOfRecord":{"articleIdentity":"rs-396764","link":"https://doi.org/10.1038/s41598-021-94659-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-07-22 15:01:53","publishedOnDateReadable":"July 22nd, 2021"},"versionCreatedAt":"2021-04-12 17:34:57","video":"","vorDoi":"10.1038/s41598-021-94659-7","vorDoiUrl":"https://doi.org/10.1038/s41598-021-94659-7","workflowStages":[]},"version":"v1","identity":"rs-396764","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-396764","identity":"rs-396764","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}