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

Abstract

The ability to perceive the bitter taste of 6-n-propylthiouracil (PROP) is a variable phenotype that has been associated with body mass index (in kg/m(2)) and linked to food choice and satiety. PROP-sensitive and -nonsensitive individuals are defined as tasters and nontasters, respectively. Sensitivity to PROP is a heritable trait based on the TAS2R38 gene on chromosome 7q34. In a recent study we demonstrated an association between PROP sensitivity and the single-nucleotide polymorphism (SNP) rs2274333 (+292A/G) within a coding sequence of the gustin/carbonic anhydrase VI gene. The purpose of this study was to develop a rapid and inexpensive screening method for identification of the rs2274333 SNP in individuals with varying sensitivity to PROP. Our results show that the methodology employed allows distinguishing A/G alleles perfectly, with a simple DNA digestion of a polymerase chain reaction fragment covering the SNP site of interest. So, the polymerase chain reaction followed by restriction fragment length polymorphism assay described in this article can be used as an alternative to sequencing in bitter taster status research, and could be employed as a survey tool in nutrigenomic studies.

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

“…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.…”

“…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.…”

“…Moreover, a recent pilot study on PROP responsiveness and thermal status was not able to confirm the association between CA6 rs2274333 and TAS2R38 polymorphism (Bering et al 2014). In light of the results obtained in the course of research on the protein CA VI (Tomassini Barbarossa et al 2011), the objective of this study was to evaluate the polymorphisms of the TAS2R38 with our new method and its interaction with CA6 genes in the modulation of the phenotype for sensitivity to PROP. Consequently, in this study, we took into consideration the TAS2R38 gene haplotypes of a group of 60 subjects with variable sensitivity to PROP and preliminarily genotyped them for the rs2274333 allele (A/G) of the CA6 gene (Tomassini Barbarossa et al 2011).…”

“…In light of the results obtained in the course of research on the protein CA VI (Tomassini Barbarossa et al 2011), the objective of this study was to evaluate the polymorphisms of the TAS2R38 with our new method and its interaction with CA6 genes in the modulation of the phenotype for sensitivity to PROP. Consequently, in this study, we took into consideration the TAS2R38 gene haplotypes of a group of 60 subjects with variable sensitivity to PROP and preliminarily genotyped them for the rs2274333 allele (A/G) of the CA6 gene (Tomassini Barbarossa et al 2011). The analysis of the nucleotide sequences upstream and downstream of the SNPs showed no presence of restriction sites for any endonuclease in the three alleles of the TAS2R38 gene (rs713598 C/G, rs1726866 C/T, and rs10246939 G/A); as such, we developed a strategy to create a restriction site artificially at each SNP for one specific enzyme.…”