Profiling of olfactory receptor gene expression in whole human olfactory mucosa

Abstract

Olfactory perception is mediated by a large array of olfactory receptor genes. The human genome contains 851 olfactory receptor gene loci. More than 50% of the loci are annotated as nonfunctional due to frame-disrupting mutations. Furthermore haplotypic missense alleles can be nonfunctional resulting from substitution of key amino acids governing protein folding or interactions with signal transduction components. Beyond their role in odor recognition, functional olfactory receptors are also required for a proper targeting of olfactory neuron axons to their corresponding glomeruli in the olfactory bulb. Therefore, we anticipate that profiling of olfactory receptor gene expression in whole human olfactory mucosa and analysis in the human population of their expression should provide an opportunity to select the frequently expressed and potentially functional olfactory receptors in view of a systematic deorphanization. To address this issue, we designed a TaqMan Low Density Array (Applied Biosystems), containing probes for 356 predicted human olfactory receptor loci to investigate their expression in whole human olfactory mucosa tissues from 26 individuals (13 women, 13 men; aged from 39 to 81 years, with an average of 67±11 years for women and 63±12 years for men). Total RNA isolation, DNase treatment, RNA integrity evaluation and reverse transcription were performed for these 26 samples. Then 384 targeted genes (including endogenous control genes and reference genes specifically expressed in olfactory epithelium for normalization purpose) were analyzed using the same real-time reverse transcription PCR platform. On average, the expression of 273 human olfactory receptor genes was observed in the 26 selected whole human olfactory mucosa analyzed, of which 90 were expressed in all 26 individuals. Most of the olfactory receptors deorphanized to date on the basis of sensitivity to known odorant molecules, which are described in the literature, were found in the expressed olfactory receptors gene set.

Figure 1. Expression profiling of candidate reference genes in whole human olfactory mucosa.

Box plot graph on C_q obtained for the reference genes specific for the olfactory epithelium across the 26 individuals samples. Left and right box limits are first and third quartiles. The inner line conventionally marks the median. Whiskers show the extreme of the series.

Figure 2. Inter-run calibration analysis.

Plotted expression pattern correlation for all detected targets (C_q average below 35) from the same RNA WHOM sample in two different runs. OR genes (green ◊), reference genes specific for the olfactory epithelium (blue Δ) and classical endogenous control genes (red ▪). C_q values above 35 are not reproducible (turquoise x). R² is the coefficient of correlation.

Figure 3. Number of OR genes expressed for each individual in function of age.

Scatterplot of the number of OR genes expressed at a level above 5 copies/20 ng RNA for each individuals. Women are colored in pink and men in blue.

Figure 4. Expression frequency of OR genes in the population of 26 individuals.

The bar chart represents the number of expressed OR genes (>5 copies/20 ng RNA) as a function of the number of expressing individuals, e.g. the number of expressed OR genes in all tested individuals (26) corresponds to 90.

Figure 5. The expression of several OR genes is statistically related with age.

22 OR genes showed a decreased expression profile and 7 OR genes showed an increased expression profile. The False discovery rate (FDR) calculated by SAM is <0.05 for all represented genes and the p value of the Spearman's correlation is <0.05 are indicated next to the name of the ORs in the heatmap.

Figure 6. Expression profiling of OR genes in whole human olfactory mucosa.

For each of the 355 OR genes (rows), the RNA copies number were estimated from normalized relative quantities obtained for each of the 26 individuals (columns). OR genes have been ranked according to their expression, from higher to lower. RNA copies number obtained for each individual are also indicated according to the green color code to show the good consistency of the inter-individual expression. OR genes with an average copies number below a cut-off of 5 copies/20 ng RNA (red arrow; right) are considered as to low or non-expressed. Age of the individuals are shown above the figure, women are colored in pink and men in blue. Published deorphanized receptors are highlighted in red on the left. Potentially non-functional OR genes are highlighted in blue on the left as well.

Figure 7. Ratio between normalized relative quantities of RNA obtained for olfactory mucosa and for inferior turbinate.

For each of the 355 OR genes, the ratio is calculated from the mean of normalized relative quantities obtained for the 26 individuals for whole human olfactory mucosa (WHOM) and from the normalized relative quantities obtained for inferior turbinate (IT; n = 1).