- Open Access
Development of a molecular method for the rapid screening and identification of the three functionally relevant polymorphisms in the human TAS2R38 receptor gene in studies of sensitivity to the bitter taste of PROP
© Orrù et al. 2015
- Received: 21 January 2015
- Accepted: 14 May 2015
- Published: 9 June 2015
The objective of this work was to develop a rapid screening method to identify the three single nucleotide polymorphisms (SNPs) in the TAS2R38 gene, with the aim of providing a significant contribution to studies designed to assess sensitivity to the bitter taste of 6-n-propylthiouracil (PROP). Specifically, the objective of this study was to characterize the TAS2R38 gene haplotypes in a group of 60 subjects with variable sensitivity to PROP and preliminarily genotyped for the rs2274333 allele (A/G) of carbonic anhydrase isoform VI gene (CA6). The molecular characterization of the TAS2R38 gene was conducted using the PCR-restriction fragment length polymorphism technique after creating artificial restriction sites upstream or downstream of the SNPs, as none of the three polymorphisms contributes to the formation of a restriction site for a specific endonuclease. The results indicate that the method described in this paper could be a valid and simple experimental strategy to identify genetic differences related to taste sensitivity to bitter taste, and could be applied as a nutrigenetics test in studies aimed at understanding people’s eating behaviors.
- Bitter taste
- PROP taster status
The experimental strategy required that each SNP be incorporated within a PCR fragment obtained with primers capable of promoting site-directed mutagenesis in the DNA, with the purpose of subsequent use in the PCR-RFLP. The primers were designed to introduce opportune nucleotide substitutions into the amplified fragments, upstream or downstream of the SNP, in order to create a region palindromic interceptable by an endonuclease. Therefore, the SNP is one of the nucleotides present in a restriction site created artificially.
Primer design strategy for the identification of polymorphism rs713598 C/G
Primer design strategy for the identification of polymorphisms rs1726866 C/T and rs10246939 G/A
To identify polymorphisms rs1726866 C/T and rs10246939 G/A, we designed a pair of primers that amplify a fragment of 194 bp (from nucleotide 739 to nucleotide 932) that contains both polymorphisms within its sequence (Figure 1). The sense primer (2F mut 5′-aagtctcttgtctcctttttctgcttctttgtgatatcatccagcg-3′) was designed by introducing a double mismatch in its sequence. Specifically, the second and fourth nucleotide starting from the 3′ end of the oligo contain, respectively, a C instead of a T and an A instead a T (as shown in the sequence). As illustrated above, these mismatches are essential for the PCR experiments, because the two T nucleotides in the sequence of the TAS2R38 gene are replaced by an A and C, respectively, in each of the amplification products. This creates the first A and the third C of the recognition sequence AGCGCT for Eco47III endonuclease, allowing the cut of the sequence only when the fragment presents the C allele (Figure 2). The reverse primer (2R mut 5′-atggtcatcacagctctcctcaacttggcattgcctgagatcagta-3′) was designed to introduce an A instead of a C in the PCR product, leading to the formation of a restriction site, GTAC, specific to the enzyme RsaI. In fact, through this modification, if the G allele typical of the super-taster is present along with the A nucleotide artificially introduced into the fragment, it leads to the formation of the first and the third nucleotide, respectively, of the cutting site recognized by the RsaI endonuclease (Figure 2). In summary, by subjecting the PCR fragment to enzymatic digestion with Eco47III, two restriction fragments of 45 and 149 bp, respectively, are generated only if the rs1726866 SNP contains the allele C characteristic of the super-taster. If the same fragment is subjected to digestion with the RsaI enzyme, the fragment is cut, even in this case, to restriction fragments of 149 and 45 bp only if the rs10246939 SNP presents the G allele characteristic of the super-taster. All primers used in PCR experiments were synthetized by Invitrogen 50 nmol scale, desalted (Europrim-Invitrogen, Cambridge, UK).
A total of 60 healthy subjects, all non-smokers, who were already genotyped for the CA6 rs2274333 SNP (Padiglia et al. 2010; Tomassini Barbarossa et al. 2011) were recruited for this research. Their mean age was 25 years, ranging 20–29 years. All of the subjects had measurable thresholds for common chemosensory stimuli and were free of medications that might affect taste or odor perception. The 60 participants were classified for PROP bitter taste status as super-tasters, medium tasters, or non-tasters using standard procedures (Tepper et al. 2001, Rankin et al. 2004). The PROP phenotype of each subject was assessed by a scaling method for taster status classification and by detection threshold measurement. The classification of subjects by taster status was determined by their PROP and NaCl ratings, using the 3-solution test. Each group had an equal number of super-tasters:medium tasters:non-tasters ratio (20:20:20). The participants were informed about the procedure and the objective of the work, and each subject reviewed and signed an informed consent form at the beginning of the protocol. The study was approved by the Ethical Committee of the University Hospital of Cagliari, Italy.
Preparation of template DNA and PCR conditions
Genomic DNA was extracted from saliva samples using the Invitrogen Charge Switch Forensic DNA Purification kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration was estimated by measurements of OD260. Purified DNA samples were stored at or below −20°C until use. The PCR reaction was carried out in a total volume of 25 μL and contained 250 ng DNA, 10 pmol of each primer, 1.5 mM MgCl2, 100 mM Tris–HCl, pH 8.3, 50 mM KCl, 200 μM of dNTP mix, and 1.5 U of Hot Master Taq Eppendorf. Reactions were performed in a Personal Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). Using pairs of 1F mut/1R primers specific for the analysis of 1° SNP (C/G) and 2F mut/2R mut specific for the analysis of the 2° and 3° SNP (C/T and G/A), we obtained amplification products of 203 and 194 bp, respectively, whose dimensions reflected those of the expected products. Amplification conditions for the 1Fmut/1R fragment consisted of initial denaturation at 95°C for 2 m, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 63°C for 30 s, and extension at 72°C for 30 s. Amplification conditions for the 2Fmut/2R fragment consisted of initial denaturation at 95°C for 2 m, followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 68°C for 30 s, and extension at 72°C for 30 s. A final extension was conducted at 72°C for 5 m for both amplification reactions. Negative controls (water instead of human DNA) were run with every PCR, and standard precautions were taken to avoid contamination.
RFLP analysis for genotyping
In this study, using mutated primers, we developed a PCR-RFLP method for the analysis of rs713598 C/G, rs1726866 C/T and rs10246939 G/A TAS2R38 SNPs. The molecular characterization of the TAS2R38 alleles was conducted after creating artificial restriction sites upstream or downstream of the SNPs of interest, as none of the three polymorphisms contributes to the formation of a restriction site for a specific endonuclease. To identify the first SNP (rs713598 C/G), the DNA was amplified with a mutated sense primer that creates, in PCR products, the first G nucleotide sequence GGCC recognition for HaeIII, thus allowing the cut of the sequence only when present in the fragment and the C allele, characteristic of taster subjects (Figure 2). In order to identify the second and third SNPs (rs1726866 C/T and rs10246939 G/A), we performed only one PCR reaction with both mutated pair primes. The sense primer was designed by introducing a double mismatch in its sequence, with the purpose of creating the first A and the third C of the recognition sequence AGCGCT for Eco47III endonuclease, allowing the cut of the sequence only when the fragment presents the C allele typical of the taster. The reverse primer was designed to introduce, in the same PCR product, the A nucleotide of the restriction site, GTAC, recognized by the RsaI enzyme. Through this modification, if the G allele typical of the taster is present along with the A nucleotide artificially introduced into the fragment, it leads to the formation of the first and the third nucleotides, respectively, of the cutting site recognized by the RsaI endonuclease (Figure 2). In order to verify the accuracy of the enzymatic digestions, all PCR samples were sequenced on both the forward and reverse strands with an ABI Prism automated sequencer. As has been widely reported in the literature, we found a match between PROP taster status and TAS2R38 gene haplotypes in the individuals studied. The statistical methods (Fisher’s method) showed, as fully described in the literature, a significant correlation (p < 0.0001), showing that the PAV haplotype is widely distributed in the general PROP tasters, while the AVI haplotype was distributed in non-tasters. As shown in Additional file 1: Table S1, and as was further observed by Calò et al. (2011), the large proportion of heterozygous PAV/AVI in the super-taster phenotype suggests that the TAS2R38 gene haplotypes justified the sensitivity to the bitter taste of PROP only in part, distinguishing only two phenotypes for the sensitivity to PROP: tasters and non-tasters. Molecular analysis of the rs2274333 (A/G) polymorphism of the CA6 gene, conducted in our previous study on the same 60 subjects, showed that the A allele and the AA genotype are more prevalent in the super-taster phenotype, while the GG genotype and the G allele are more prevalent in the non-taster phenotype (Tomassini Barbarossa et al. 2011). In view of the results obtained in the present study, we correlated the TAS2R38 gene polymorphisms with the rs2274333 (A/G) SNP of the CA6 gene in the three groups of subjects. According to the results obtained by Calò et al. (2011), a high percentage of PAV/AVI subjects, observed after restriction analysis and validated by sequencing data, can justify a super-taster phenotype when this heterozygous status is associated with the A allele of the rs2274333 CA6 polymorphism (Additional file 2: Table S2). Thus, the high sensitivity to PROP in the Sardinian population super-tasters seems to be determined by the association (p value <0.0001, calculated using the Markov Chain method) of gene haplotype PAV TAS2R38 with at least one A allele of the CA6 gene.
Even if the mutation does not result in a restriction site difference, it was possible to exploit the difference between the two allelic forms of each SNP of TAS2R38 gene, by amplification-created restriction site PCR. Thus, by using the PCR-RFLP technique developed in our laboratory, we established a simple, efficient and low-cost method that can be used in any laboratory equipped with basic instrumentation for the study of DNA, to determine the allelic forms of the TAS2R38 gene. Some of the important advantages of the PCR-RFLP technique include inexpensiveness and the lack of need for advanced instrumentation or extensive training of laboratory staff. Not all research laboratories have a DNA sequencer or else a real-time instrument to observe the SNPs by labeled primers or expensive TaqMan-type probes. Thus, such laboratories must send their samples to outside laboratories that are equipped with specific instrumentation. Some of the disadvantages of the PCR-RFLP technique could be that this experimental approach requires relatively large amounts of hands-on time when compared to other techniques such as direct sequencing. With regard to genotyping by sequencing, to obtain the nucleotide sequence it is, however, obligatory to have subjected the DNA to a PCR reaction. Also, the time employed for digestion of the PCR products using the PCR-RFLP method may also be the same as the time required for sequencing, especially if the rapid digestion restriction enzymes are used. Thus, the method described above, validated by direct sequencing, could be useful to reconstruct TAS2R38 gene haplotypes, and might represent an experimental strategy to identify differences in gustatory sensibility. Since taste perception plays a key role in determining individual food preferences and dietary habits, the method could be applied as a test of nutrigenetics in studies aimed at understanding eating behaviors. Until recently, TAS2R38 has been considered the only gene involved in gustatory sensibility to the bitter taste, but the data obtained in this study confirm, in a Sardinian genetic isolate, that TAS2R38 haplotypes and the polymorphisms rs2274333 (A/G) of the carbonic anhydrase VI gene might cooperate in the modulation of the phenotype related to sensitivity to PROP. Genetic differences in genes involved in taste perception between ethnic groups may contribute to differences in patterns of diet. Understanding these differences in genetic variations may help to explain ethnic differences in the risk for chronic diseases, and could lead to the development of appropriate public health measures of prevention.
AP and RO designed the study. EA and RO carried out PCR-RFLP experiments, data acquisition and the literature review. AP drafted the manuscript. All authors read and approved the final manuscript.
This study was partially supported by CAR funds from Cagliari University and by a grant from “Fondazione Banco di Sardegna”. The authors thank the volunteers, without whose contribution this study would not have been possible to carry out. We also thank Iole Tomassini Barbarossa (University of Cagliari, Cagliari, Italy) for the bitter taste analysis with PROP and Francesca Manchinu (CNR Neurogenetics and Neuropharmacology Institute, Cagliari, Italy), for sequencing the polymerase chain reaction products.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Avenet P, Lindemann B (1989) Perspectives of taste reception. J Membr Biol 112:1–8View ArticleGoogle Scholar
- Baranowski T, Baranowski JC, Watson KB, Jago R, Islam N, Beltran A et al (2011) 6-n-propylthiouracil taster status not related to reported cruciferous vegetable intake among ethnically diverse children. Nutr Res 31:594–600. doi:10.1016/j.nutres.2011.07.004 View ArticleGoogle Scholar
- Tomassini Barbarossa I, Atzori E, Zonza A, Padiglia A (2011) A rapid screening method for the identification of a single-nucleotide polymorphism in the carbonic anhydrase VI gene in studies of sensitivity to the bitter taste of 6-n-propylthiouracil. Genet Test Mol Biomark 15:721–724. doi:10.1089/gtmb.2011.0040 View ArticleGoogle Scholar
- Bartoshuk LM (2000) Comparing sensory experiences across individuals: recent psychophysical advances illuminate genetic variation in taste perception. Chem Senses 25:447–460. doi:10.1093/chemse/25.4.447 View ArticleGoogle Scholar
- Behrens M, Meyerhof W (2006) Bitter taste receptors and human bitter taste perception. Cell Mol Life Sci 63:1501–1509. doi:10.1007/s00018-006-6113-8 View ArticleGoogle Scholar
- Bell KI, Tepper BJ (2006) Short-term vegetable intake by young children classified by 6-n-propylthoiuracil bitter-taste phenotype. Am J Clin Nutr 84:245–251Google Scholar
- Bering AB, Pickering G, Liang P (2014) TAS2R38 single nucleotide polymorphisms are associated with prop—but not thermal—tasting: a pilot study. Chem Percept 7:23–30. doi:10.1007/s12078-013-9160-1 View ArticleGoogle Scholar
- Bufe B, Breslin PA, Kuhn C, Reed DR, Tharp CD, Slack JP et al (2005) The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception. Curr Biol 15:322–327. doi:10.1016/j.cub.2005.01.047 View ArticleGoogle Scholar
- Calò C, Padiglia A, Zonza A, Corrias L, Contu P, Tepper BJ et al (2011) Polymorphisms in TAS2R38 and the taste bud trophic factor, gustin gene co-operate in modulating PROP taste phenotype. Physiol Behav 104:1065–1071. doi:10.1016/j.physbeh.2011.06.013 View ArticleGoogle Scholar
- Dinehart ME, Hayes JE, Bartoshuk LM, Lanier SL, Duffy VB (2006) Bitter taste markers explain variability in vegetable sweetness, bitterness, and intake. Physiol Behav 87:304–313. doi:10.1016/j.physbeh.2005.10.018 View ArticleGoogle Scholar
- Drayna D, Coon H, Kim UK, Elsner T, Cromer K, Otterud B et al (2003) Genetic analysis of a complex trait in the Utah Genetic Reference Project: a major locus for PTC taste ability on chromosome 7q and a secondary locus on chromosome 16p. Hum Genet 112:567–572. doi:10.1007/s00439-003-0911-y Google Scholar
- Drewnowski A, Henderson SA, Cockroft JE (2007) Genetic sensitivity to 6-n-propylthiouracil has no influence on dietary patterns, body mass indexes, or plasma lipid profiles of women. J Am Diet Assoc 107:1340–1348. doi:10.1016/j.jada.2007.05.013 View ArticleGoogle Scholar
- Duffy VB, Bartoshuk LM, Lucchina LA, Snyder DJ, Tym A (1996) Supertasters of PROP (6-n-propylthiouracil) rate the highest creaminess to high-fat milk products. Chem Senses 21:598. doi:10.1093/chemse/21.5.573 Google Scholar
- Duffy VB, Davidson AC, Kidd JR, Kidd KK, Speed WC, Pakstis AJ et al (2004) Bitter receptor gene (TAS2R38), 6-n-propylthiouracil (PROP) bitterness and alcohol intake. Alcohol Clin Exp Res 28(11):1629–1637. doi:10.1097/01.ALC.0000145789.55183.D4 View ArticleGoogle Scholar
- Feeney EL, Hayes JE (2014) Exploring associations between taste perception, oral anatomy and polymorphisms in the carbonic anhydrase (gustin) gene CA6. Physiol Behav 128:148–154. doi:10.1016/j.physbeh.2014.02.013 View ArticleGoogle Scholar
- Genick UK, Kutalik Z, Ledda M, Destito MC, Souza MM, Cirillo CA et al (2011) Sensitivity of genome-wide-association signals to phenotyping strategy: the PROP-TAS2R38 taste association as a benchmark. PLoS One 6:e27745. doi:10.1371/journal.pone.0027745 View ArticleGoogle Scholar
- Hamilton RB, Norgren R (1984) Central projections of gustatory nerves in the rat. J Comp Neurol 222:560–577. doi:10.1002/cne.902220408 View ArticleGoogle Scholar
- Hayes JE, Duffy VB (2007) Revisiting sugar-fat mixtures: sweetness and creaminess vary with phenotypic markers of oral sensation. Chem Senses 32:225–236. doi:10.1093/chemse/bjl050 View ArticleGoogle Scholar
- Herring A, Inglis N, Ojeh C, Snodgrass D, Merizies J (1982) Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels. J Clin Microbiol 16:473–477Google Scholar
- Horn T, Castilho L, Moulds JM, Billingsley K, Vege S, Johnson N et al (2012) A novel JKA allele, nt561C >A, associated with silencing of Kidd expression. Transfusion 52:1092–1096. doi:10.1111/j.1537-2995.2011.03399.x View ArticleGoogle Scholar
- Jiang L, Yin X, Yin C, Zhou S, Dan W, Sun X (2011) Different quantitative EEG alterations induced by TBI among patients with different APOE genotypes. Neurosci Lett 505:160–164. doi:10.1016/j.neulet.2011.10.011 View ArticleGoogle Scholar
- Keller KL, Steinmann L, Nurse RJ, Tepper BJ (2002) Genetic taste sensitivity to 6-n-prophylthiouracil influences food preference and reported food intake in preschool children. Appetite 38:3–12. doi:10.1006/appe.2001.0441 View ArticleGoogle Scholar
- Kim UK, Drayna D (2004) Genetics of individual differences in bitter taste perception: lessons from the PTC gene. Clin Genet 67:275–280. doi:10.1111/j.1399-0004.2004.00361.x View ArticleGoogle Scholar
- Kim UK, Jorgenson E, Coon H, Leppert M, Risch N, Drayna D (2003) Positional cloning of the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide. Science 299:1221–1225. doi:10.1126/science.1080190 View ArticleGoogle Scholar
- Lindemann B (1996) Taste reception. Physiol Rev 76:718–766Google Scholar
- Liu Y, Huang L, Lu Y, Xi XE, Huang XL, Lu Q et al (2015) Relationships between the osteocalcin gene polymorphisms, serum osteocalcin levels, and hepatitis B virus-related hepatocellular carcinoma in a chinese population. PLoS One 10:e0116479. doi:10.1371/journal.pone.0116479 View ArticleGoogle Scholar
- Margolskee RF (2001) Molecular mechanisms of bitter and sweet taste transduction. JBC 277:1–4. doi:10.1074/jbc.R100054200 View ArticleGoogle Scholar
- Melis M, Atzori E, Cabras S, Zonza A, Calò C, Muroni P et al (2013) The gustin (ca6) gene polymorphism, rs2274333 (A/G), as a mechanistic link between prop tasting and fungiform taste papilla density and maintenance. PLoS One 8:e74151. doi:10.1371/journal.pone.0074151 View ArticleGoogle Scholar
- Padiglia A, Zonza A, Atzori E, Chillotti C, Calò C, Tepper BJ et al (2010) Sensitivity to 6-n-propylthiouracil is associated with gustin (carbonic anhydrase VI) gene polymorphism, salivary zinc, and body mass index in humans. Am J Clin Nutr 92:539–545. doi:10.3945/ajcn.2010.29418 View ArticleGoogle Scholar
- Prodi DA, Drayna D, Forabosco P, Palmas MA, Maestrale GB, Piras D et al (2004) Bitter Taste Study in a sardinian genetic isolate supports the association of phenylthiocarbamide sensitivity to the TAS2R38 bitter receptor gene. Chem Senses 29:697–702. doi:10.1093/chemse/bjh074 View ArticleGoogle Scholar
- Rankin KM, Godinot N, Christensen CM, Tepper BJ, Kirkmeyer SV (2004) Assessment of different methods for 6-n-propylthiouracil status classification. In: Prescott J, Tepper BJ (eds) Genetic variation in taste sensitivity. Marcel Dekker, New York, pp 63–88. doi: 10.1201/9780203023433.ch3
- Robino A, Mezzavilla M, Pirastu N, Dognini M, Tepper BJ, Gasparini P (2014) A population-based approach to study the impact of PROP perception on food liking in populations along the silk road. PLoS One 9:e91716. doi:10.1371/journal.pone.0091716 View ArticleGoogle Scholar
- Sambrook J, Fritstch E, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
- Tepper BJ (2008) Nutritional implications of genetic taste variation: the role of PROP sensitivity and other taste phenotypes. Annu Rev Nutr 28:367–388. doi:10.1146/annurev.nutr.28.061807.155458 View ArticleGoogle Scholar
- Tepper BJ, Nurse RJ (1998) PROP taster status is related to fat perception and preference. Ann N Y Acad Sci 855:802–804. doi:10.1111/j.1749-6632.1998.tb10662.x View ArticleGoogle Scholar
- Tepper BJ, Cao J, Christensen CM (2001) Development of brief methods to classify individuals by PROP taster status. Physiol Behav 73:571–577. doi:10.1016/S0031-9384(01)00500-5 View ArticleGoogle Scholar
- Timpson NJ, Christensen M, Lawlor DA (2005) TAS2R38 (phenylthiocarbamide) haplotypes, coronary heart disease traits, and eating behaviour in the British Women’s Heart and Health Study. Am J Clin Nutr 81:1005–1011Google Scholar
- Ueda T, Ugawa S, Yamamura H, Imaizumi Y, Shimada S (2003) Functional interaction between T2R taste receptors and g-protein subunits expressed in taste receptor cells. J Neurosci 23:7376–7380Google Scholar
- Wiener A, Shudler M, Levit A, Niv MY (2012) BitterDB: a database of bitter compounds. Nucleic Acids Res 40(Database issue):D413–D419. doi:10.1093/nar/gkr755 View ArticleGoogle Scholar
- Wooding S, Kim UK, Bamshad MJ, Larsen J, Jorde LB, Drayna D (2004) Natural selection and molecular evolution in PTC, a bitter-taste receptor gene. Am J Hum Genet 74:637–646. doi:10.1086/383092 View ArticleGoogle Scholar