Reluciferase assay system. Measurements revealed a detection threshold of 1 mM and an EC50 value of 116 mM for the humanHuman TAAR5 Is Activated by TrimethylamineTable 1. SNPs in the coding regions of hTAAR genes.SNP Position Gene TAAR1 SNP ID rs8192620 rs8192619 TAAR2 rs61745666 rs8192646 TAAR5 rs3813355 rs3813354 TAAR6 rs8192622 rs8192624 rs8192625 TAAR8 TAAR9 rs8192627 rs2842899 rs9402420 on Chr. 6 132966279 132966348 132938817 132938842 132910612 132910634 132891538 132892253 132892332 132874814 132859609 132859437 in mRNA 864 795 528 503 266 244 78 793 872 983 181Nucleotide Reference T G G C G C C G G A A C SNP C A A T A T T A A C T TSNP Frequency ESP Cohort 22.9 5.4 1.9 2.2 65.2 9.4 4.9 8.0 6.8 7.2 27.0 9.2 Ctrl Pool 30.2 4.7 3.1 5.6 66.2 10.8 6.4 6.7 6.5 5.8 24.6 6.5 TMA Anos. 28.6 7.1 0.0 0.0 71.4 7.1 0.0 7.1 14.3 0.0 35.7 0.0 p-value (FET) 1.000 0.506 1.000 1.000 0.781 1.000 1.000 1.000 0.243 1.000 0.351 1.Calculation of Fisher’s Exact Test based on SNP percentage difference between control pool and TMA anosmics. doi:10.1371/journal.pone.0054950.tTAAR5, while the EC50 for murine TAAR5 was previously reported to be nearly 400-fold lower (300 nM) [2]. Due to the different assay sensitivities the comparability of the EC50 values is somehow limited. Therefore, we tested the murine TAAR5 Epigenetics receptor activity and determined an EC50 value of 940 nM under the same Cre-luciferase assay conditions we used for human TAAR5 (Figure S2). This confirms that the murine TAAR5 is more sensitive than the human ortholog, at least in a recombinant system. However, it could still play an important role within human inhibitor olfaction. In a recombinant system the co-expression of different proteins like REEPs or RTPs can influence the receptor cell-surface expression [14,29], which essentially determines measured intensities of receptor activation. We co-transfected RTP1S and Golf that might increase the surface expression of m/hTAAR5 and general assay sensitivity, but there might be even more optimized expression conditions for each receptor. It is also possible that receptors expressed in vivo in OSNs are more sensitive than receptors expressed in vitro in a recombinant system. The olfactory detection threshold for TMA in water is 4.761027 g/l, 
which is equivalent to 8 nM [30]. In a recombinant system, even the sensitive murine TAAR5 is not activated by such a low TMA concentration. The low olfactory detection 
threshold for TMA is similar to that for 5a-androst-16-en-3-one, a human steroid in male and female urine and sweat [30]. In vitro, the olfactory receptor OR7D4 is selectively activated by androstenone with an EC50 value of 12 mM, which is also above the olfactory threshold concentration [31]. It seems to be not quite clear to what extent receptor sensitivities in recombinant 1527786 systems can be transferred to in vivo situations, where the receptor is expressed in native OSNs. Nevertheless, the general functionality can be tested. Furthermore, there is a link between the function of OR7D4 in vitro and the rating of androstenone in vivo [31], as well as between the function of OR11H7P in vitro and threshold variations in the perception of isovaleric acid in vivo [32]. In both cases, SNPs in the coding sequence of odorant receptors were responsible for phenotypic variations. Many odor-specific anosmias are known, although their molecular background remains enigmatic. Thus, we investigated whether any SNP in a functional hTAAR gene was associated with TMA anosmi.Reluciferase assay system. Measurements revealed a detection threshold of 1 mM and an EC50 value of 116 mM for the humanHuman TAAR5 Is Activated by TrimethylamineTable 1. SNPs in the coding regions of hTAAR genes.SNP Position Gene TAAR1 SNP ID rs8192620 rs8192619 TAAR2 rs61745666 rs8192646 TAAR5 rs3813355 rs3813354 TAAR6 rs8192622 rs8192624 rs8192625 TAAR8 TAAR9 rs8192627 rs2842899 rs9402420 on Chr. 6 132966279 132966348 132938817 132938842 132910612 132910634 132891538 132892253 132892332 132874814 132859609 132859437 in mRNA 864 795 528 503 266 244 78 793 872 983 181Nucleotide Reference T G G C G C C G G A A C SNP C A A T A T T A A C T TSNP Frequency ESP Cohort 22.9 5.4 1.9 2.2 65.2 9.4 4.9 8.0 6.8 7.2 27.0 9.2 Ctrl Pool 30.2 4.7 3.1 5.6 66.2 10.8 6.4 6.7 6.5 5.8 24.6 6.5 TMA Anos. 28.6 7.1 0.0 0.0 71.4 7.1 0.0 7.1 14.3 0.0 35.7 0.0 p-value (FET) 1.000 0.506 1.000 1.000 0.781 1.000 1.000 1.000 0.243 1.000 0.351 1.Calculation of Fisher’s Exact Test based on SNP percentage difference between control pool and TMA anosmics. doi:10.1371/journal.pone.0054950.tTAAR5, while the EC50 for murine TAAR5 was previously reported to be nearly 400-fold lower (300 nM) [2]. Due to the different assay sensitivities the comparability of the EC50 values is somehow limited. Therefore, we tested the murine TAAR5 receptor activity and determined an EC50 value of 940 nM under the same Cre-luciferase assay conditions we used for human TAAR5 (Figure S2). This confirms that the murine TAAR5 is more sensitive than the human ortholog, at least in a recombinant system. However, it could still play an important role within human olfaction. In a recombinant system the co-expression of different proteins like REEPs or RTPs can influence the receptor cell-surface expression [14,29], which essentially determines measured intensities of receptor activation. We co-transfected RTP1S and Golf that might increase the surface expression of m/hTAAR5 and general assay sensitivity, but there might be even more optimized expression conditions for each receptor. It is also possible that receptors expressed in vivo in OSNs are more sensitive than receptors expressed in vitro in a recombinant system. The olfactory detection threshold for TMA in water is 4.761027 g/l, which is equivalent to 8 nM [30]. In a recombinant system, even the sensitive murine TAAR5 is not activated by such a low TMA concentration. The low olfactory detection threshold for TMA is similar to that for 5a-androst-16-en-3-one, a human steroid in male and female urine and sweat [30]. In vitro, the olfactory receptor OR7D4 is selectively activated by androstenone with an EC50 value of 12 mM, which is also above the olfactory threshold concentration [31]. It seems to be not quite clear to what extent receptor sensitivities in recombinant 1527786 systems can be transferred to in vivo situations, where the receptor is expressed in native OSNs. Nevertheless, the general functionality can be tested. Furthermore, there is a link between the function of OR7D4 in vitro and the rating of androstenone in vivo [31], as well as between the function of OR11H7P in vitro and threshold variations in the perception of isovaleric acid in vivo [32]. In both cases, SNPs in the coding sequence of odorant receptors were responsible for phenotypic variations. Many odor-specific anosmias are known, although their molecular background remains enigmatic. Thus, we investigated whether any SNP in a functional hTAAR gene was associated with TMA anosmi.
