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Peer-reviewed

Research Article

Profiling of Olfactory Receptor Gene Expression in Whole Man Olfactory Mucosa

  • Christophe Verbeurgt,
  • Françoise Wilkin,
  • Maxime Tarabichi,
  • Françoise Gregoire,
  • Jacques E. Dumont,
  • Pierre Chatelain

PLOS

ten

  • Published: May half dozen, 2014
  • https://doi.org/ten.1371/journal.pone.0096333

Abstract

Olfactory perception is mediated past a large array of olfactory receptor genes. The human genome contains 851 olfactory receptor gene loci. More than l% of the loci are annotated every bit nonfunctional due to frame-disrupting mutations. Furthermore haplotypic missense alleles can be nonfunctional resulting from substitution of cardinal amino acids governing protein folding or interactions with signal transduction components. Across their function in smell recognition, functional olfactory receptors are as well required for a proper targeting of olfactory neuron axons to their corresponding glomeruli in the olfactory bulb. Therefore, nosotros conceptualize that profiling of olfactory receptor gene expression in whole human being olfactory mucosa and assay in the human population of their expression should provide an opportunity to select the ofttimes expressed and potentially functional olfactory receptors in view of a systematic deorphanization. To accost this event, nosotros designed a TaqMan Low Density Array (Applied Biosystems), containing probes for 356 predicted human olfactory receptor loci to investigate their expression in whole homo olfactory mucosa tissues from 26 individuals (13 women, xiii 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 contrary transcription were performed for these 26 samples. And so 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 being olfactory mucosa analyzed, of which xc were expressed in all 26 individuals. Nearly of the olfactory receptors deorphanized to engagement on the basis of sensitivity to known odorant molecules, which are described in the literature, were establish in the expressed olfactory receptors gene set.

Citation: Verbeurgt C, Wilkin F, Tarabichi Chiliad, Gregoire F, Dumont JE, Chatelain P (2014) Profiling of Olfactory Receptor Gene Expression in Whole Homo Olfactory Mucosa. PLoS ONE 9(five): e96333. https://doi.org/x.1371/journal.pone.0096333

Editor: Richard David Newcomb, Institute and Nutrient Research, New Zealand

Received: Jan xv, 2014; Accepted: April 7, 2014; Published: May half-dozen, 2014

Copyright: © 2014 Verbeurgt et al. This is an open-access article distributed nether the terms of the Creative Commons Attribution License, which permits unrestricted utilise, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Thousand. Tarabichi is supported past FRIA/FNRS grant. URL: http://www.fnrs.exist/. C. Verbeurgt is supported by Fonds Erasme, Université Libre de Bruxelles, Brussels, Belgium. URL: http://world wide web.fondserasme.org/fonds-erasme-pour-la-recherche-medicale. ChemCom Due south.A. is supported by Innoviris (Brussels Found for Research and Innovation) URL: http://www.innoviris.exist/site/. The funders had no office in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: Two authors are employed by a commercial company (ChemCom S.A.): P. Chatelain and F. Wilkin. The authors have declared that no competing interests exist and this does not modify our adherence to PLOS ONE policies on sharing data and materials.

Introduction

Analysis of published mammalian genomes indicates that olfactory receptor (OR) genes constitute by far the largest gene family. Initially, Buck and Axel identified this extremely large multigene family based on the observation that OR genes were expressed in olfactory epithelium of rat [1]. Later, other members of this family unit were identified by sequence homology with the first fix of OR genes [2]–[4]. Currently information technology is accepted that the human being genome contains 851 OR loci. More than 50% of the loci are annotated as nonfunctional due to frame-disrupting mutations, leaving approximately 400 potentially functional OR genes.

In spite of this rather accurate genomic characterization, very little is known of the functional and integrative mechanisms of human being olfactory receptor in odorant perception. To appointment, the responses of just 48 human ORs with one or more than odorant molecules have been reported [v]–[xx] and less than ten of these receptors have been reliably associated with olfactory perception of an odorant stimuli [8]–[10], [xiii], [fourteen].

In the quest to develop industrial applications based on the employ of man odorant receptors, ChemCom is committed to the systematic identification of ligands for these chemoreceptors. To fulfill this aggressive deorphanization project, because the huge number of anticipated functional OR genes, it is mandatory to obtain clues about the involvement of the targeted ORs in the olfactory perception. Moreover, the expression of several predicted OR genes has been detected in non-olfactory tissues, suggesting that a subset of predicted OR genes could take functions unrelated to olfaction. Indeed, expression of OR transcripts has been described in various tissues, including testis and spermatozoa [19], [21]–[26], prostate [27]–[30], enterochromaffin cells [6], pulmonary neuroendocrine cells [31], brain [32]–[35], tongue [36]–[38], erythroid cells [39], placenta [40], breast [41] and kidney [42]. In improver, systematic expression profiling of ORs in non-olfactory tissues using EST data, microarray or deep sequencing analysis [43]–[45] have shown that a large number of putative human being OR genes are expressed in these tissues. The analysis of the unabridged olfactory subtranscriptome in a variety of different human tissues provides a listing of several OR genes that are highly expressed in non-olfactory tissues [44]. At least some of these ORs could play a role in spermatozoa chemotactism [19], in musculus regeneration [46] or in claret pressure level regulation [47]. Although, it cannot exist excluded that OR may nowadays double olfactory and non-olfactory functions; it remains possible that some members of the reported odorant receptors family could be solely non-olfactory G protein-coupled receptors.

Another issue pertaining to ORs deorphanization results from the significant allelic variation observed for human ORs. A recent data mining of the sequence repository of the m Genomes Project, has estimated that the number of variants per OR locus is on average about ten. Nevertheless, some variants may be nonfunctional missense haplotypic alleles [48]. Furthermore, as it has been demonstrated in mice that functional olfactory receptors are required for proper targeting of olfactory neuron axons to their corresponding glomeruli in the olfactory bulb [49], one may suppose that alleles of OR genes predominantly expressed in the olfactory epithelium stand for to functional haplotypes.

Taken together, a written report of OR factor expression in the whole human olfactory mucosa (WHOM) provides an opportunity to define ORs specifically involved in olfaction, allowing choosing frequently expressed and potentially functional ORs for deorphanization campaigns. OR gene expression in WHOM has been seldomly studied, probably due to the difficult access to human material. Two publications have reported the characterization of the expression of the human OR factor family unit in 3 individuals merely using Dna microarray and just in i individual using deep sequencing [45], [l]. Therefore, we designed an innovative approach based on a TaqMan Low Density Array (TLDA) containing probes for 356 predicted OR loci to investigate more thoroughly the OR cistron expression profile in human olfactory mucosa. Real-time reverse transcription PCR (qRT-PCR) is frequently used for gene expression quantification, at the transcriptional level, due to its reproducibility and sensitivity. The method has besides go the preferred method for validating results obtained by other techniques, such as microarrays or deep sequencing.

Herein nosotros nowadays our information obtained using an innovative loftier throughput transcriptome profiling approach of human OR genes, in WHOM of much larger gear up of 26 individuals.

Materials and Methods

Ethics argument

This project was approved by the Erasme Infirmary ideals committee (ULB, Brussels, Belgium: P2011/135 and A2013/050).

Patients and tissues specimens

WHOM were collected 27±12 hours mail-mortem from 26 individuals (thirteen women and xiii men; aged from 39 to 81 years, with an boilerplate of 67±11 years for women and 63±12 years for men). Most individuals were of European origin. For each patient, the clinical data is summarized in Table 1. Patients with a history of dysosmia or rhinologic diseases, including allergic rhinitis and chronic sinusitis, were excluded. We also investigated the history of smoking, associated with smell'southward disorder probably related to alterations of the olfactory mucosa [51], [52]. Amid the 26 subjects, 8 were smokers and the smoking status was unknown for 4 of them.

The WHOM was accurately dissected from the septum, the cribriform plate, the eye and the superior turbinates. As the boundaries between the olfactory and the respiratory epithelium are not conspicuously defined in humans [53], the septal mucosa was dissected upwardly to the lower limit of the eye turbinate. A control tissue was taken from the mucosa of the inferior turbinate. The dissected tissue samples (nigh 3.5×5 cm from each side of the olfactory cleft mucosa) were collected, frozen in liquid nitrogen and stored immediately at −80°C.

Total RNA isolation

Frozen WHOM was crushed in liquid nitrogen. Total RNA was purified and treated with DNase using the RNeasy kit (Qiagen) according to manufacturer instructions. DNase treatment is mandatory as intron spanning primers is non possible due to lack of introns in the OR genes. Quantitative and qualitative assessment of RNA samples (pooled from each side) was performed by NanoDrop spectophotometry (Thermo Scientific) and by microfluidic analysis using a 2100 Bioanalyser (Agilent Technologies). This latter technique produces an electropherogram allowing the evaluation of the integrity of the 18S and 28S ribosomal RNAs (Figure S1A and S1B) and the algorithm assigns a RNA integrity number (RIN) ranging from 1 to 10, where 10 corresponds to ideally intact RNA and 1 to highly degraded RNA (Table one).

cDNA synthesis

Full RNA (1 µg per sample-loading port of the 48 PCR reaction channels) was used in the reverse transcription (RT) Quantiscript reaction (Qiagen), performed with a combination of oligo-dT primer and random hexamers following the manufacturer'due south protocol. Each RNA sample was additionally run on one port (feeding 48 PCR assays) of the TaqMan Low Density Assortment (TLDA) in the absence of reverse transcriptase (RT-) to assess its potential contamination by genomic DNA. The latter, resulted for all samples, in a borderline amplification for a small-scale subset of the big panel of intronless genes tested. On average, 90% of the PCR yielded a quantification bicycle (Cq) value labeled as undetermined or above 35 cycles. The remaining ten% gave an boilerplate Cq of 34.ane±one.1 indicating a potential low residuum genomic Deoxyribonucleic acid contamination.

TLDA pattern and training

A customized TLDA was designed in collaboration with Applied Biosystems. The design process for the assays is described in the White Paper TaqMan Gene Expression Assays from Practical Biosystems. The software TaqExpress was used for the design. Whenever possible, the assays were designed to dilate part of the gene coding sequence. The context sequence determines gauge assay position and the assay IDs allows retrieving the details from Practical Biosystems website (Tabular array S1).

The 384 wells of the TLDA comprise FAM dye-labeled NFQ probes and primers for an internal control (GAPDH, 4 wells), 10 endogenous control genes exhibiting low differential expression across tissues (MRPL19, CASC3, POLR2A, CDKN1B, TBP, RPL30, PSMC4, YWHAZ, UBC, PPIA), 356 human OR genes, and vi reference genes specifically expressed in olfactory epithelium (CNGA2, GNAL, ADCY3, RIC8B, RTP1, OBP2A&2B) [50], [54]. cDNA (pre-mixed with TaqMan Universal PCR Master Mix) was loaded onto the TLDA and PCR amplifications were performed in a 7900HT Thermocycler (Applied Biosystems). Thermal cycling conditions used were: 2 min at 50°C, 10 min at 94.5°C, followed by 40 cycles at 97°C for 30 sec, and 59.7°C for 1 min.

Existent-fourth dimension PCR with genomic DNA

One TLDA carte was run with 150 ng (per port) of a pooled human being genomic Deoxyribonucleic acid from Clontech to evaluate the efficacy of the assays.

Real-fourth dimension PCR with plasmid Dna

I TLDA card was run with 30 pg (per port) of a pool of 30 OR coding plasmids cloned by ChemCom to evaluate the specificity of the assays. The receptors chosen to perform this experiment were spread throughout the different families of OR genes represented past an unrooted tree based on similarity of amino acid properties. One pg of each plasmid represents almost 3000 molecules of specific plasmid per PCR.

TLDA analysis and Statistical assay

The real-time PCR focuses on the exponential phase, where amplification doubles target templates, following the exponential amplification (2due north where north is the number of cycles). The real-time PCR instrument calculates a Cq value representing the PCR cycle at which the reaction reaches a fluorescent intensity threshold above background. For Cq calculation, the threshold was manually set at ΔRn = 0.one for all samples and all targets (threshold set inside the twodue north exponential distension phase). The results were analyzed using the Sequence Detection Systems (SDS) version 2.iv and Qbase+ software packages [55]. Conclusion of the optimal number of reference genes for the normalization of qPCR data was performed using the geNorm algorithm [56].

Association analyses were performed with R 2.14.1 [R Evolution Core Team (2008). R: A linguistic communication and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Republic of austria. ISBN iii-900051-07-0, URL http://www.R-project.org.]. Significance Analysis of Microarray (SAM) [http://www.pnas.org.gate1.inist.fr/content/98/nine/5116.total] was performed using the samr parcel v2.0 Genes with q-value below 0.05 were considered pregnant. For each variable (i.due east. age, sexual practice and smoking status) SAM was performed to find the receptors presenting expressions individually associated with each variable. To assess whether the expressions of all receptors were globally associated with i of these variables, the sum of the square of scores of association (Pearson correlation coefficients for historic period, t-scores for the two other variables) of all the receptors expressions was compared to a cypher-distribution of the sum of the scores of associations obtained after x.000 permutations of the patient labels. Heatmap visualization was obtained with the heatmap.2 function inside the gplots v2.ten.1 parcel [gplots: Diverse R programming tools for plotting information (2011), Gregory R. Warnes, URL http://CRAN.R-project.org/package=gplots].

Results

A 384-customized TLDA was designed to investigate the gene expression of a big array of OR genes from 26 WHOM samples. All experiments were performed according to the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines [57].

Assay of RNA purity and integrity

The 260/280 and 260/230 OD ratios were measured for all RNA samples to appraise respectively the purity of RNA with respect to protein contagion and residual organic solvent. All samples used showed a 260/280 and 260/230 OD ratios betwixt 1.8 and ii.0, indicative of proficient quality RNA with minimal contaminations. RNA integrity was also assessed, and samples characterized by RIN (RNA integrity number) ranging from 5.nine to nine.0 were used, the average ±SD being seven.3±0.eight (Figure S1A, S1B, Table 1). These RIN values are ordinarily considered adequate for qRT-PCR experiments [58]. No correlation between the RIN and the delay betwixt death and sample collection was observed (Statistical assay reveal a p value = 0.36 for a Pearson'south correlation with a Rii = 0.035).

Option of candidate reference genes

Classical endogenous control genes, exhibiting minimal differential expression across unlike tissues (MRPL19, CASC3, POLR2A, CDKN1B, TBP, RPL30, PSMC4, YWHAZ, UBC, PPIA) were added to the TLDA, in order to perform a first technical normalization and to compare expression of genes from different tissues as for instance WHOM and inferior turbinate. The structure of WHOM is often patchy and contains a meaning proportion of respiratory epithelium [54], [59]. Therefore, to compare expression of genes from dissimilar WHOM, assays for tissue specific olfactory epithelium reference genes were too added to the TLDA for a second biological normalization purpose. The half dozen selected genes were CNGA2, GNAL, ADCY3, RIC8B, RTP1 and OBP2A&2B.

Expression profiling and stability assay of candidate reference genes

Boilerplate Cq values of classical endogenous reference genes was 17.1±0.7 for UBC (hateful ± SD), 17.6±0.5 for GAPDH, 18.0±0.six for PPIA, 20.0±0.vi for CDKN1B, xx.four±0.half-dozen for CASC3, 21.5±0.vii for PSMC4, 21.7±0.6 for POLR2A, 22.1±0.8 for YWHAZ, 22.six±0.ix for RPL30, 22.eight±iii.6 for MRPL19 and 24±0.6 for TBP (Tabular array S1). Their stabilities were evaluated by the geNorm algorithm [56] and the geometric hateful of 3 stably expressed classical endogenous reference genes (CASC3, PSMC4, CDKN1B) were selected to technically normalize the results. Cq of the olfactory epithelium-specific reference genes (Ric8B, GNAL, RTP1, CNGA2, ADCY3, OBP) are shown in the box plots of Effigy one. The distribution of the olfactory epithelium-specific reference genes Cq provides a global representation of the variation of reference factor expression likewise equally information on their relative abundance. More than highly expressed genes are associated with lower Cq. Boilerplate Cq values ranged from 21.iii±0.8 (ADCY3) to thirty.6±1.1 (OBP, ways ±SD, northward = 26) (Table S1). As suggested in Khan et al. [54], in view of obtaining a biologically relevant normalization, the geometric hateful of the six specific reference genes provided a normalizing cistron, rather than a gene from a single reference gene.

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Effigy 1. Expression profiling of candidate reference genes in whole man olfactory mucosa.

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

https://doi.org/10.1371/journal.pone.0096333.g001

Real-time PCR with genomic Dna

One TLDA card was run with a pool of human genomic DNA to evaluate the assays (individual gene efficiency amplification). The Cq average value of 351 detected OR genes is 24.five±0.8 (Table S2) suggesting similar amplification rates for all the genes, as expected equally the number of targets is identical for all genes in a human genome. Furthermore 21 assays gave an expected undetermined Cq values considering they correspond either to reference genes or to 4 OR genes for which the assays are designed with intron spanning primers. These assays are identified by a suffix '_m' in the assayID. One out of 4 GAPDH assays gave a not-expected value of 36 and PPIA gave a not-expected value of 27 whereas these assays are designed with intron spanning primers. This reflects a slight non-meaning amplification compared with results obtained on RNA (delta-Cq of 18 for GAPDH and 9 for PPIA). Finally, one target (OR2A14) showed an abnormal Cq value of 12.4, therefore this assays has not been taken into business relationship for the qRT-PCR analysis.

Existent-fourth dimension PCR with plasmid Dna

I TLDA card was run with a pool of thirty OR coding plasmids to evaluate the specificity of the assays. The expected specific PCR amplification of the 30 targets gives an average Cq value of 25.4±1.ane, (mean ±SD, n = thirty). However, we observe a not-specific distension for 15 additional receptors. For xi of them, the average Cq value is 32.5±1.8, (mean ±SD, due north = 11) which reflects a delta-Cq value of 7 as compared to specific amplification. In this case, the non-specific amplification is so negligible. For four of them, the boilerplate Cq value is 25.0±0.viii, (hateful ±SD, north = four) which reflects no difference as compared to specific amplification. These iv couples of genes OR2L3 and OR2L8 (97% identity on the entire DNA sequence), OR52E6 and OR52E8 (90% identity), OR52I1 and OR52I2 (97% identity) and OR5D16 and OR5D18 (83% identity) cannot be discriminated by this TLDA card. Then for the thirty OR coding plasmids, 88% of the PCR amplifications are specific for the target.

Inter-run scale

3 different experiments were conducted to run the 26 samples, to correct for possible run-to-run variation whenever all samples are not analyzed in the same run, identical sample take been tested in all runs. Figure 2 shows the correlation between Cq values for all detected targets (ie. Cq boilerplate <35) from the same ARN sample in ii dissimilar runs. Cq values above 35 are not reliable considering duplicates are not reproducible. The correlation coefficient reaches 0.83 and the intercept is 0.91 for the detected OR genes. The correlation coefficient reaches 0.99 for the olfactory epithelium-specific reference genes and for classical endogenous control genes.

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Figure 2. Inter-run calibration analysis.

Plotted expression blueprint correlation for all detected targets (Cq average below 35) from the same RNA WHOM sample in two different runs. OR genes (dark-green ◊), reference genes specific for the olfactory epithelium (blueish Δ) and classical endogenous control genes (carmine ▪). Cq values above 35 are non reproducible (turquoise x). R2 is the coefficient of correlation.

https://doi.org/ten.1371/journal.pone.0096333.g002

Expression profiling of olfactory receptors genes in WHOM

On average, for the 26 samples, Cq values computed from amplification plots of 355 OR genes range between 25.8 and 39.eight (Table S1). These results reflect a low expression of the OR genes compared to other genes involved in the olfactory pour. One target (OR2A14) shows an abnormal amplification plot with a Cq value of xvi.6±7.3 (hateful ±SD); this assays will not be taken into business relationship for the analysis. On boilerplate, 62±29 (hateful ±SD) OR genes per sample gave an undetermined Cq value which was arbitrarily assigned to 40 cycles to allow the calculation of an boilerplate Cq values. 74±34 (mean ±SD) OR genes per sample gave a Cq value above 35 were considered as expressed at very low level or not expressed at all.

To brand a more quantitative analysis, the Cq values of each OR were converted into normalized relative quantities (NRQ) following the method previously described [55]. Briefly, we apply the delta-Cq quantification model using the average Cq obtained for all ORs in the 26 individuals every bit calibrator (here 32.seven) which is transformed into relative quantities using the exponential function, so results are fully equivalent and thus simply rescaled. Then the normalization of relative quantities was performed with the geometric hateful of the multiple stably expressed classical endogenous reference genes (CASC3, PSMC4, CDKN1B) defined by the geNorm algorithm [56] and followed by the normalization with the geometric hateful of the vi reference genes specific for olfactory epithelium. Results obtained on genomic Dna and on specific OR coding plasmids allowed to calculate the approximate number of copies of target for twenty ng RNA engaged in the RT-PCR reaction (Table S3).

For each private, the detected OR factor number (>5 copies/xx ng RNA) was counted (Effigy iii). This cut-off value corresponds to a Cq value ≥35.three. On average, on the 355 OR gene targets, the detected OR gene number is 232±28 for women and 238±28 for men.

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Figure three. 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 pinkish and men in blue.

https://doi.org/ten.1371/journal.pone.0096333.g003

At that place is a substantial divergence in the expressed OR gene repertoire of each of the samples. A set of ninety human OR genes were detected (>5 copies/20 ng RNA) in all tested individuals. Another set of 140 human being OR genes were detected not in all tested individuals but in more than half of the population (in 13 individuals and more on 26) and a third set composed of 125 human OR genes were more rarely detected (in less than 13 individuals on 26) (Effigy 4).

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

The bar chart represents the number of expressed OR genes (>five copies/20 ng RNA) equally a office of the number of expressing individuals, e.1000. the number of expressed OR genes in all tested individuals (26) corresponds to 90.

https://doi.org/ten.1371/periodical.pone.0096333.g004

Globally, the OR cistron expression was not associated with age (p value = 0.xix), sex (p value = 0.23) or smoking (p value = 0.66, Pearson's correlation). Individually, 22 OR genes showed a decreased profile and 7 OR genes showed an increased profile related with historic period (Figure 5). There is no significant clan betwixt individual OR cistron expression and sex or smoking.

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Effigy 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'south correlation is <0.05 are indicated next to the name of the ORs in the heatmap.

https://doi.org/10.1371/journal.pone.0096333.g005

Effigy 6 shows OR genes ranked in office of their expression level, from the highest to lowest. Information technology shows 273 (77%) human OR genes in a higher place the cutting-off value of 5 copies/20 ng RNA. No significant enrichment in grade I or grade II ORs is observed in the expressed set. Indeed, 17.6% of OR genes expressed belong to class I while 15.2% of the OR genes tested belong to this class.

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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 accept been ranked according to their expression, from higher to lower. RNA copies number obtained for each individual are too indicated co-ordinate to the light-green color lawmaking to show the good consistency of the inter-individual expression. OR genes with an boilerplate copies number below a cutting-off of five copies/twenty ng RNA (cerise arrow; correct) 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 every bit well.

https://doi.org/10.1371/periodical.pone.0096333.g006

Interestingly, most of the published deorphanized olfactory receptors [5]–[11], [13]–[20] are plant into the prepare of expressed OR genes (Figure 6). Indeed, nosotros count 43 expressed OR genes among the 47 deorphanized receptors described in the literature which are tested in this report. In other words, xvi% of expressed OR genes are deorphanized while this percentage drops down to 4.8% for non-expressed OR genes (p value = 0.009, Fisher's exact test).

An changed distribution is observed with potentially non-functional OR genes. Expression levels of 52 OR genes with mutations affecting positions in the consensus amino acid motifs specific for OR genes [60] were analyzed. These receptors, although regarded as intact OR genes, harbor a mutation affecting P or Y in the LHT P Yard Y motif, or affecting M, R or the 2nd A in the Chiliad AYD R YV A IC motif, or Y in the Due south Y motif or finally, on H in the FSTCSS H motif. All known variants of these OR genes, stand for to a mutated haplotype of these highly conserved positions [48]. Presumably, these receptors are no longer functional (highlighted in blueish in the Figure half-dozen). We observed that 25% of non-expressed OR genes are potentially not-functional whereas eleven.three% from the expressed fix are potentially not-functional (p value = 0.002, Fisher's exact test). In addition, the average RNA copy numbers of the 47 deorphanized receptors (174±247) and of the 52 potentially non-functional ORs (48±115) are significantly different (p value = 0.002, Student's t-Examination, two-tailed distribution, two-samples with unequal variance).

Equally shown in Table 2, the average RNA copy number varies drastically among the 47 reported deorphanized receptors. The most expressed OR gene corresponds to OR7C1 with an estimate average re-create number of well-nigh 1108/20 ng RNA. A huge divergence has also been noted in the expression of the 4 closely related paralogs, OR10G3, OR10G4, OR10G7 and OR10G9 that respond to ethyl vanillin and eugenol [5]. Indeed OR10G3 is well expressed in 25/26 WHOM samples with an boilerplate of 487 copies/twenty ng RNA. OR10G4 and OR10G7 are moderately expressed (with an average of 29 and 13 copies/20 ng RNA respectively) and OR10G9 is detected merely in 3 WHOM samples above the cut-off of v copies (with an average of two copies/20 ng RNA). We can count 19 ORs expressed by the 26 individuals among the 47 genes. In others words, 21% of the group of ninety receptors expressed in every individual were deorphanized. The four non-expressed ORs (OR1A2, OR2M7, OR5D18, OR10G9) are expressed simply in i, 4, 0 and iii individuals respectively.

Expression profiling of olfactory receptor genes in junior turbinate sample

One sample of inferior turbinate (It) has been tested in a TLDA and gives 210 undetermined Cq values compared to 62 on average in WHOM tissues. This observation reflects the not-detection of OR cistron expression, expected for a non-olfactory tissue. To make a more quantitative analysis, the Cq values were converted into normalized relative quantities with classical endogenous stably expressed reference genes (CASC3, PSMC4, CDKN1B) defined by the geNorm algorithm. Figure 7 shows results obtained for OR gene expression ratio betwixt WHOM and junior turbinate. We notice 250 OR genes (70%) more expressed in WHOM than in inferior turbinate (ratio WHOM/Information technology≥2) (Table S4). Some reference genes specific for olfactory sensory neurons (CNGA2 and Ric8B) are significantly expressed more than in WHOM than in IT; RTP1 and ADCY3 are expressed a little more than in WHOM than in IT while OBP and GNAL are detected in equivalent amount in both tissues.

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Figure 7. Ratio betwixt normalized relative quantities of RNA obtained for olfactory mucosa and for junior turbinate.

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

https://doi.org/10.1371/periodical.pone.0096333.g007

Word

Although the OR cistron family was discovered over twenty years agone by Buck and Axel, few information are bachelor on their expression in human olfactory mucosa, contrasting with the recent pregnant increase of results on the genetic polymorphism of OR genes [48]. This probably reflects the difficulty to learn human being tissue and obtain good quality RNA from WHOM.

We report here the get-go extensive loftier throughput transcriptome profiling of OR cistron expression directly by real-time contrary transcription PCR performed furthermore on WHOM from a relatively large population of 26 patients. Indeed, our study was focused on the expression of 356 predicted functional OR genes amid the 851 OR loci scattered throughout the human genome.

But a small per centum of the olfactory mucosa consists of olfactory sensory neurons. Moreover, the boundaries of the WHOM are unclear and the tissue is sometimes replaced by respiratory epithelium. Furthermore, the fraction of olfactory sensory neurons can vary significantly from i sample to some other. Therefore, to allow OR cistron expression comparisons between individual WHOM samples, normalization is mandatory. Consequently, we therefore normalized our expression data from the individual WHOM, with 6 and so called tissue specific reference genes, that are expressed specifically by olfactory sensory neurons as previously described by Khan et al. [54].

Our results show that 77% of human being intact OR gene repertoire are expressed with an average level above 5 copies/20 ng RNA. A set up of xc man OR messengers were detected in all tested individuals. In addition, lxx% of the human OR gene repertoire were found more expressed in WHOM than in the inferior turbinate (ratio WHOM/Information technology ≥2). Along with the widespread genetic variation reported for man OR protein coding regions, that correlates to individual differences in odorous perception [8], [9], [13], [14] and with genetic variations in auxiliary olfactory genes [50], this differential expression of ORs in the WHOM could plant the basis to a well-documented inter-individual variation in olfactory sensitivity.

Statistical assay was performed for each clinical variable (i.east. age, sexual activity and smoking status) to appraise if receptor expressions were globally or individually associated. Our results indicate that OR gene expression is globally not associated with age, sex activity or smoking status. Withal, we are non able to find if these clinical factors may reduce the absolute amount of olfactory sensory neurons. As we have normalized our data with specific references genes of olfactory sensory neurons, nosotros have not taken into account the absolute amount of olfactory tissue. Therefore, the OR cistron expression is described relatively to olfactory sensory neurons. Furthermore, at this bespeak, our results cannot explicate the decrease of olfactory performance related to age or smoking [51], [52], [61].

Nevertheless, our results show that individually, the expression of 22 OR genes seems to subtract significantly with age, and the expression of 7 OR genes seems to increase significantly. These results tin be compared to those of Khan et al. [54] where the majority of OR gene expression (58.4%) in mice remained stable during aging, while 32.8% presented downwardly profiles, seven.2% upward profiles and i.7% of convex or concave profiles. We institute no correlation between individual OR gene expression and sex or smoking, although some clinical observations show that these two conditions may influence smell abilities. These differences in smell abilities may occur at another level of the olfactory organisation than the OR gene expression.

Prior to the nowadays work, only two studies had focused on the expression of the man OR factor family. In these studies, Deoxyribonucleic acid microarray [45] or deep sequencing [fifty] were used every bit experimental approaches. Latter written report focused on accessory proteins and presented results in the supplementary data merely for one human being olfactory epithelium biopsy. Over the 261 intact ORs overlapping with our study, 174 OR genes were found to be expressed in this olfactory epithelium biopsy (threshold set at a FPKM≥0.i) whereas we found 202 OR genes expressed in the WHOM (threshold prepare at a number of copies ≥5). 145 genes (i.e.72% of our expressed OR gene set) turned out to be common to both studies and 30 ORs are not expressed in WHOM co-ordinate to both studies (Table S5). Therefore, our approach is in understanding with the previous report past Keydar et al. apropos expressed OR genes (p value = 0.0012, hypergeometric exam) and for the expression levels of each OR genes (p value<0.001, Spearman'southward correlation) [fifty]. With respect to the Zhang et al. written report [45], at that place are 319 intact ORs overlapping with our study. 202 OR genes were establish to exist more expressed in human olfactory epithelium than in other tissues by Zhang et al. (threshold set at a p value<0.01) whereas we constitute 225 OR genes preferentially expressed in WHOM (threshold set at a ratio WHOM/It≥2). Furthermore, 142 genes (i.east. 63% of our expressed OR factor set) turned out to exist common to both studies and 34 ORs are not expressed in WHOM according to both studies (Table S6). The overlap of 63% is exactly what would be expected if the two datasets are completely random with respect to each other (p value = 0.iii, hypergeometric test). Moreover, no correlation betwixt the expression levels of each OR genes could be observed between the two studies (p value = 0.96, Spearman'southward correlation). It is noteworthy that the non-olfactory tissues used to estimate a divergence in gene expression are not the same. We compared the expression in the WHOM to the one of inferior turbinate, while homo liver, lung, kidney, eye and testis were used in the study by Zhang et al. The latter therefore must be taken with caution, equally non-olfactory expression of ORs have been reported for these unlike tissues, and could therefore bias the comparative results they obtained. We tried to exclude the set of non-olfactory expressed ORs from our comparison. Even excluding these receptors, we could not reveal a correlation between the current data and the Zhang et al. data. Some other source of discrepancy between both studies relies on the proportion of the olfactory epithelium used to determine OR expression. In the publication past Zhang et al., it is not clear whether the analyzed tissues cover the entire olfactory mucosa or only a determined anatomical section. This difference might be highly significant as the distribution of olfactory receptors is not homogeneous in the olfactory epithelium of rodents [62], [63]. Importantly, though no data currently be in humans. Some other difficulty relies on the fact that the boundaries betwixt the olfactory and the respiratory epithelium are not clearly defined in humans. Different publications report that the human olfactory epithelium is located on the nasal septum, the cribriform plate, the superior and the centre turbinate [53], [64], [65]. Consequently, this motivated united states collecting all these anatomical regions. The mucosa of the nasal septum was dissected along the projection of the middle turbinate. Thus, our samples correspond practically the totality of the olfactory mucosa. Finally, our results are in good agreement with those of Keydar et al., but not with Zhang et al. Moreover, we constitute no correlation between Keydar et al. and Zhang et al. (p value = 0.341, hypergeometric test and p value = 0.13, Spearman's correlation).

Interestingly, we discover an enrichment of functional deorphanized receptors in the set of expressed OR genes and an enrichment of potentially non-functional receptors into the set of non-expressed OR genes. This corroborates the observations of Zhang et al. The latter reports that lxxx% of intact OR genes and 67% of OR pseudogenes were found to be expressed in WHOM and moreover intact ORs appear to be expressed at a higher level on average than OR pseudogenes.

Taken together, these observations support the hypothesis predicting that if a gene is expressed, it is more than likely to be functional. Indeed, a non-functional OR can lead to a defective targeting of olfactory sensory neurons in the olfactory bulb and therefore reduces the survival of these neurons [66]. More than precisely, the proper targeting seems more related to OR-derived campsite signals rather than the OR ability to demark an odorant [67]. The relation between OR genes functionality and expression could be further explored by studying the variants of expressed OR genes. Indeed, for known ORs already deorphanized, both functional and not-functional haplotypes have been described; consequently, it would exist worth determining whether expressed allelic variants correspond preferentially to functional haplotypes.

A systematic report of OR gene expression profiles, expressed in non-olfactory tissues using deep sequencing analysis has been recently reported and provides a listing of highly expressed OR genes [44]. From this list, 32 intact OR genes are mutual with our study. We confirmed that the bulk of the non-olfactory tissues expressed OR genes (28/32; 87%) are likewise expressed in WHOM (more than than five copies/twenty ng RNA). 24 out of 32 (75%) non-olfactory tissues expressed OR genes are expressed more than in WHOM than in inferior turbinate (ratio WHOM/IT ≥two). The remaining viii OR genes are detected similarly in both tissues (0.96< ratio WHOM/IT1 <ii). From this comparing, it does not seem that non-olfactory tissues expressed OR genes make up a separate grouping, with a putative not-olfactory role, that would clearly segregate from ORs expressed in the WHOM. Yet, for one particular receptor, OR2W1, we observed a very low expression level in the WHOM whereas it is well detected in pulmonary neuroendocrine cells [31]. Upon its deorphanization, this receptor was establish accept a broad spectrum of stimuli [fifteen]. This OR is activated past more than than 200 molecules and interacts with a large variety of chemical structures eliciting very different odors [Veithen et al., unpublished information]. Together, our results and those of Gu et al. [31] suggest a function for OR2W1 in the detection of volatile irritants in the human being airways. Therefore, this receptor may offer an example of an OR family member that would really not be merely an olfactory mucosa odorant receptor.

Although our study represents the most all-encompassing analysis of human OR expression in the olfactory mucosa, it does present some limitations. The considered population is relatively onetime. Indeed, because of the difficulty to obtain human material, samples from patients presenting characteristics that may bear on the olfactory mucosa such as age and smoking, take not been discarded. However, these conditions do not appear to change the OR genes expression. On some other mitt, our study includes most exclusively subjects of European origin and therefore does non explore the possible ethnic related variations of OR gene expression. Logically, therefore, we acknowledge that it would exist worth extending the analysis to samples from other origins, if available.

All the same these points and since the majority of human being olfactory receptors are non deorphanized, the information on the expression of OR genes in WHOM nerveless in this study offers an essential preliminary and lacking understanding that will allow focusing future research on frequently expressed and potentially functional olfactory receptors identified.

Supporting Information

Figure S1.

Analysis of RNA integrity. A. Electropherogram showing the integrity of 9 homo olfactory epithelium RNA samples (lanes 1 to 9). B. Example of profile showing a RIN of 8.five (sample 8, RNA from a woman of 72 years old) and the integrity of the 18S and 28S ribosomal RNA.

https://doi.org/ten.1371/journal.pone.0096333.s001

(TIF)

Table S5.

Comparison between the 2 studies by Verbeurgt et al. and by Keydar et al.: listing of 145 ORs that are expressed in the WHOM according to both studies and list of thirty ORs that are non expressed in the WHOM according to both studies.

https://doi.org/x.1371/journal.pone.0096333.s006

(XLSX)

Table S6.

Comparison between the 2 studies by Verbeurgt et al. and by Zhang et al.: listing of 142 ORs that are expressed in the WHOM according to both studies and list of 34 ORs that are non expressed in the WHOM according to both studies.

https://doi.org/10.1371/journal.pone.0096333.s007

(XLSX)

Acknowledgments

The authors thank J. Perret from the Laboratory of Pathophysiological and Nutritional Biochemistry, Department of Biochemistry, Free University of Brussels, Brussels, Kingdom of belgium, for discussions on q-RTPCR and experimental pattern, use of the Qbase+ Software, help in analyzing information and manuscript critical reviewing as well equally for spelling and grammer. We give thanks C. Degraef and F. Libert from the Institute of Interdisciplinary Inquiry in human and molecular Biological science, Free University of Brussels, Brussels, Belgium, for technical aid and fruitful discussions respectively. We also give thanks A. Veithen and S. Patiny from ChemCom S.A., Brussels, Kingdom of belgium for valuable comments on the manuscript.

Author Contributions

Conceived and designed the experiments: CV FW FG JED PC. Performed the experiments: CV FW. Analyzed the data: CV FW MT FG. Contributed reagents/materials/assay tools: CV FW MT FG. Wrote the paper: CV FW MT FG JED PC.

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