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Is there variation in which olfactory receptor genes are functional in humans?

Is there variation in which olfactory receptor genes are functional in humans?


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According to wikipedia humans have around 400 functional olfactory receptor genes and 600 pseudo genes. Most mammals have higher numbers of functional OR genes.

My question is whether it has been studied how the number or subset of these functional ORs varies between humans.


Perhaps the most dramatic example of variation in olfactory abilities in humans is anosmia, or the inability to smell. However, this isn't always due to broken olfactory receptors -- some total anosmias are due to cilia (small hairs) that don't function properly. (This paper mentions that.)

Specific anosmias, or insensitivities to particular smells, are probably closer to what you're asking about. The textbook chapter I linked to below talks about how proportions of the population lack the ability to smell certain odorants -- for example, about 1/10 people cannot detect ethyl mercaptan, which is the scent given to natural gas leaks. These specific anosmias are generally restricted to just one odorant (smell), which suggests that it is likely a receptor deficit. (EDIT: The comments below have even more examples of scents that vary from person to person.)

Interestingly, many conditions are associated with a decline in smelling ability and range, like Alzheimers, diabetes, and taking certain medications. These declines can be because of fewer olfactory receptors or because of deficits further along in sensory processing.

I couldn't find any papers assessing smell variation across all humans -- doctors rarely think to assess smell, so there isn't much data on "normal" populations. Also, it doesn't seem like the genes controlling olfactory receptors in humans have been characterized well, so it would be extremely difficult to do a comparison on the genotypic level.

In sum, there are definitely differences in the ability to smell a wide range of scents. To my knowledge, this hasn't been characterized in an especially systematic way. (If it has, I hope somebody on here knows otherwise!)

This chapter of Purves' Neuroscience gives a basic run-down of olfaction and some possible variations in it. https://www.ncbi.nlm.nih.gov/books/NBK11032/


There's a gene for detecting that fishy smell, olfactory GWAS shows

For many people, the smell of fish is rather strong and unpleasant. But some people carry a mutation in a particular gene that makes that fish odor less intense, reports a paper publishing October 8 in the journal Current Biology. The study, which is the largest genome-wide association study (GWAS) of olfactory genes in humans involving a sniff test and looked at over 9,000 people from Iceland, also shows that people vary in their ability to discern the smell of licorice and cinnamon.

"We discovered sequence variants that influence how we perceive and describe fish, licorice, and cinnamon odors," said Rosa Gisladottir of deCODE Genetics in Reykjavik, Iceland. "Since our sense of smell is very important for the perception of flavor, these variants likely influence whether we like food containing these odors."

Researchers have known that people perceive odors based on olfactory receptors encoded by 855 olfactory genes. But about half of those genes in people are thought to lack function, leaving us with a relatively small repertoire of about 400 olfactory genes. The reason humans have lost so many olfactory genes has remained mysterious. It is also not well understood how variation in these genes might influence differences among people in their sense of smell.

To explore this in the new study, Gisladottir and her colleagues including Kari Stefansson, also of deCODE, enlisted 9,122 Icelanders in a GWAS in search of variants that influence odor perception. To do it, they asked study participants to smell odors presented to them in pen-like devices that released a particular scent when uncapped. After sniffing each odor "pen," the researchers asked them to name the smell. Participants also rated the intensity and pleasantness of the smell. Those odors included key ingredients found in licorice, cinnamon, fish, lemon, peppermint, and banana.

Their search turned up variants in three genes or genetic loci of interest, which they were able to confirm in a separate sample of 2,204 Icelanders. One of them is in a non-canonical olfactory receptor gene called trace amine-associated receptor 5 (TAAR5). The TAAR5 variant affects perception of fish odor containing trimethylamine, a compound found in rotten and fermented fish, as well as other animal odors and various bodily secretions. In the smell tests, people with a particular variant of this gene were more likely to not smell anything when presented with the fish odor or to use descriptors for it that were neutral or positive and not seafood related, such as "potatoes," "caramel," and "rose." The findings are the first to show an important role for this gene in people, the researchers say.

"Carriers of the variant find the fish odor less intense, less unpleasant, and are less likely to name it accurately," Gisladottir said. "There is a lot of animal research on TAAR5 in relation to its role in hard-wired aversive responses to trimethylamine. Our findings extend the implications of this research to human odor perception and behavior."

The other two discoveries were found in more typical and common olfactory gene variants. They influenced an individual's ability to name licorice and cinnamon odors. They also influenced the intensity and pleasantness associated with those odors.

"We discovered a common variant in a cluster of olfactory receptors which is associated with increased sensitivity to trans-anethole, found in black licorice products but also in spices and plants such as anise seed, star anise, and fennel," Gisladottir said. "Carriers of the variant find the licorice odor more intense, more pleasant, and can name it more accurately. Interestingly, the variant is much more common in East Asia than in Europe."

The cinnamon variant influenced the perception of trans-cinnamaldehyde, the major ingredient in both Chinese and Ceylon cinnamon. Carriers of the variant can name the cinnamon odor more accurately, they report. They also find it more intense.

Overall, the findings show that variation in olfactory genes influences odor perception in humans. They also show that, while humans have fewer olfactory genes compared to other species, some of the genetic variation that people do carry makes them more sensitive to particular smells such as licorice or cinnamon, not less.

"When coupled with evidence for geographical differences in allele frequencies, this raises the possibility that the portion of the extensive sequence diversity found in human olfactory receptor genes that affects our sense of smell is still being honed by natural selection," the researchers wrote.

The researchers say they will continue to collect data on odor perception in people. They also plan to use the same olfactory tasks to investigate smell deficits in the context of COVID-19.

This work was supported in part by the Swedish Society for Medical Research. Several authors are employees of deCODE genetics/Amgen, Inc.

Current Biology, Gisladottir et al.: "Sequence variants in TAAR5 and other loci affect human odor perception and naming" https://www.cell.com/current-biology/fulltext/S0960-9822(20)31343-9

Current Biology (@CurrentBiology), published by Cell Press, is a bimonthly journal that features papers across all areas of biology. Current Biology strives to foster communication across fields of biology, both by publishing important findings of general interest and through highly accessible front matter for non-specialists. Visit: http://www. cell. com/ current-biology. To receive Cell Press media alerts, contact [email protected]

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.


OR genes in humans

The entire set of OR genes present in the human genome was determined by several groups[9–11]. By conducting extensive homology searches,

800 OR genes were identified, but, interestingly, more than half of them were found to be pseudogenes (Figure 1)[11]. Therefore, the number of functional genes in humans is < 400. Here, the distinction between a functional gene and a pseudogene was made on the basis of its sequence. When an intact coding sequence could be recovered with an initiation codon and a stop codon in the proper positions, such a sequence was considered to be functional, while a sequence disrupted by nonsense or frameshift mutations or long deletions was regarded as a pseudogene. OR genes form many genomic clusters and are scattered all over the human genome, with the exceptions of chromosome 20 and the Y chromosome[11]. In particular, chromosome 11 contains > 40 per cent of all OR genes. The genes located close to each other on a chromosome tend to be evolutionarily closely related, suggesting that the number of OR genes has increased by repeated tandem gene duplications[11]. The relationships between genomic clusters and evolutionary kinships, however, are often complicated by chromosomal rearrangements[11]. For example, it is known that human chromosomes 14 and 15 were generated by chromosome fission in the common ancestor of great apes, and that the fission event occurred at a cluster of OR genes[15]. Nevertheless, the organisation of OR gene clusters are generally well conserved between humans and mice, and orthologous relationships can easily be identified between the two species[16]. The number of OR genes in mice (

1,000 Figure 1) is much higher than that in humans, and thus each genomic cluster in mice contains a larger number of OR genes than in humans, on average. This observation suggests that the greater OR gene repertoires in mice relative to humans have been generated mainly by gene duplications within each cluster.[16]

Numbers of OR genes in 23 chordate species[12–14]. F, T and P indicate the numbers of functional genes, truncated genes and pseudogenes, respectively. A truncated gene is part of an intact sequence that is located at a contig end. The fraction of pseudogenes (P per cent) in each species was estimated by assuming that all truncated genes are functional[12]. Phylogenetic relationships among the 23 species are also shown.

Each OR is thought to be specialised to recognise physicochemical features of odour molecules, such as functional groups or molecular size however, the relationships between ORs and odour molecules are largely unknown. Recently, Saito et al.[17] conducted high-throughput screening of 93 different odours against 464 ORs expressed in heterologous cells and succeeded in identifying ligands for 52 mouse and ten human ORs. Yet, so far, ligands have been identified for only

100 mammalian ORs. Mammalian OR genes are known to be classified into two groups, class I and class II, according to sequence similarity[18]. The functional difference between class I and class II genes is still unclear, but it has been hypothesised that class I and class II genes are for detecting relatively hydrophilic and hydrophobic compounds, respectively.[19]

Human olfactory perception differs largely among individuals. For example, one individual in ten cannot perceive hydrogen cyanide, an extreme poisonous gas[20]. One in 1,000 does not smell butyl mercaptan, the odour of the skunk. Such phenomena are called specific anosmia, meaning specific loss or impairment of smell[20]. Another example is androstenone, a pig pheromone. This odour is perceived as offensive ('sweaty, urinous'), pleasant ('sweet, floral'), or odourless. Recently, Keller et al.[21] revealed that the genetic variation in a human OR gene, OR7D4, which is selectively activated by androstenone, accounts for the variation in the perception of androstenone. There is a common variant of this receptor containing two amino acid substitutions, and homozygous or heterozygous subjects with these amino acid changes are less sensitive and have less unpleasant perceptions of androstenone.

It is also known that OR genes are highly polymorphic in humans. It has been reported that > 60 OR loci are segregating pseudogenes, in which both an intact allele and a pseudogenised allele exist in the human population[22]. Menashe et al.[23] conducted a genome-wide association study and found a significant association between the presence of a nonsense single nucleotide polymorphism (SNP) in an OR, OR11H7P, and detection threshold differences for the sweaty odorant, isovaleric acid. Nozawa et al.[24] examined copy number variations in human OR genes and suggested that the difference in the number of functional OR genes between two individuals is


Discussion

It is common ground that olfactory sensitivity differs across individuals, and in some cases, this feature has been related to genetic variations. Thus, the contribution of the genotype in the perception of odorants and volatile chemical mixtures seems particularly relevant. The highly diverse ORs, at the membrane of the olfactory neurons, trigger the first input of the olfactory signal. Thus, genomic studies of this family of receptors represent an important source of knowledge for academics and industry professionals who study human olfaction. To this end, we can take advantage of the vast amount of information on natural genetic variations coming from the genome-data community shared initiatives freely available in the public domain.

Using data mining tools, close to 120,000 nucleotide variations in human ORs were obtained from the large-scale sequencing data repository gnomAD, which provides well-structured information of sequencing data from a wide variety of sequencing projects all over the world [21]. The curation and computer analysis of this variation data revealed an uneven distribution of mutations in OR genes, reflecting the active role of natural selection in this family of receptors. Moreover, a considerable proportion of the identified mutations occur at very low frequencies, many of them uniquely identified at definite ethnic groups or individuals. This extraordinary genotypic variation has been earlier described [59] and suggests a great phenotypic diversity in the olfactory perception between humans.

The striking variation in the OR gene repertoire has motivated their study and characterization by computational methods for several years [60]. These tools have been fundamental in the identification of inactive members of the family (e.g., the Classifier for Olfactory Receptor Pseudogenes (CORP) algorithm [14]), as well as for exploring the olfactory repertoires (e.g., the Olfactory Receptors Database (ORDB) [61] and the Human Olfactory Data Explorer (HORDE) [62]). Nevertheless, more progress is required in the development of new data analysis interfaces that facilitate the integration of OR information with structural knowledge. Taking into account the increasing need for tools providing accurate predictions of functional consequences of natural variants identified in genomic studies [63] evolutionary conservation and structural context were considered as key elements in the estimation of the functional role of the natural variations identified. It is worth stressing that, in many cases, the structural framework of the mutated sites (intimately linked to the stability, function, and interactions) is often overlooked due to a limited structural knowledge [64]. ORs are not an exception to this reality, with no molecular structure reported to date. However, the highly conserved molecular architecture and sequence motifs that characterize the class A GPCR family make it possible to reliably predict the topological positions of the identified mutations from structure-informed sequence alignments. Using this approach, we provide a 3D context for the many variants occurring in ORs facilitating the functional interpretation of the changes attending to their structural location, biochemical associated data, and substitution score weightings. This method is exemplified through the identification of several natural OR variants located at conserved topological sites (e.g., BW 2.50, 3.50, 7.50, 45.50 at ECL2), either involved in the structural stability or in the functional mechanism of the receptors, and which might induce changes in the odorant sensitivity.

We believe the integration of high-throughput sequencing data with structural information is crucial for the interpretation of the complex genotype-phenotype associations occurring not only in human olfaction, but also in any other biological process. These would require in many cases the development of automatic interfaces to facilitate the management and organization of large quantities of data. Hence, we developed an interactive computational application that integrates both genomic and structural knowledge with analytical graphical tools for the study of the OR mutational landscape. The human Olfactory Receptor Mutation Database (hORMdb) allows the comparison, topological localization, and evaluation of natural variations occurring in human ORs, and represents to our knowledge, one of the largest collections of variation data of human sensory proteins annotated at the structural level.


Unravelling the complexity of human olfactory receptor repertoire by copy number analysis across population using high resolution arrays

Olfactory receptors (OR), responsible for detection of odor molecules, belong to the largest family of genes and are highly polymorphic in nature having distinct polymorphisms associated with specific regions around the globe. Since there are no reports on the presence of copy number variations in OR repertoire of Indian population, the present investigation in 43 Indians along with 270 HapMap and 31 Tibetan samples was undertaken to study genome variability and evolution. Analysis was performed using Affymetrix Genome-Wide Human SNP Array 6.0 chip, Affymterix CytoScan(®) High-Density array, HD-CNV, and MAFFT program. We observed a total of 1527 OR genes in 503 CNV events from 81.3% of the study group, which includes 67.6% duplications and 32.4% deletions encompassing more of genes than pseudogenes. We report human genotypic variation in functional OR repertoire size across populations and it was found that the combinatorial effect of both "orthologous obtained from closely related species" and "paralogous derived sequences" provide the complexity to the continuously occurring OR CNVs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The first, second and third…

Figure 1. The first, second and third bars in groups A–D represent Indian, HapMap and…

Figure 2. Karyogram indicating the CNPs in…

Figure 2. Karyogram indicating the CNPs in different clusters of Indian, Tibetan and Hapmap OR…

Figure 3. Venn diagrams representing the number…

Figure 3. Venn diagrams representing the number of overlapping common Copy Number Polymorphisms (CNPs) found…

Figure 4. A Heat Map of Log…

Figure 4. A Heat Map of Log R ratios indicating the quantitative assessments of genotyping…

Figure 5. Hot spot detection on OR…

Figure 5. Hot spot detection on OR CNPs was identified using HD-CNV software which generated…

Figure 6. Divergence of CNV across genome.

Figure 6. Divergence of CNV across genome.

The tree shows the divergence of CNVs present…

Figure 7. Phylogenetic tree of the flanking…

Figure 7. Phylogenetic tree of the flanking recombining upstream and downstream sequence breakpoints of CNV…

Figure 8. Network of genes involved in…

Figure 8. Network of genes involved in olfactory perception with hub genes distributed in three…


A Nasty Niff or Passably Pleasant ? Study Finds Genetic Variation in How We Perceive Fishy Smells

Through what they claim is the largest genome-wide association study (GWAS) of olfactory genes, researchers in Iceland have identified genetic variants that affect how people perceive the strength and unpleasantness of fish odor. Many people find the smell of fish strong and unpleasant, but the new study—which involved more than 9,000 people in Iceland, and a sniff test—showed how some people carry a mutation in a particular gene that makes fish odor less intense. The researchers also identified other gene mutations that impact on the ability to discern the smell of licorice, and cinnamon.

“We discovered sequence variants that influence how we perceive and describe fish, licorice, and cinnamon odors,” said Rosa Gisladottir, PhD, from deCODE Genetics and the University of Iceland, who is first and co-corresponding author of the team’s published paper in Current Biology. “Since our sense of smell is very important for the perception of flavor, these variants likely influence whether we like food containing these odors.” Gisladottir and colleagues who are affiliated with universities in Iceland and in Sweden, reported on their findings in a paper titled, “Sequence Variants in TAAR5 and Other Loci Affect Human Odor Perception and Naming.”

Human olfaction is critical for a variety of functions and provides us with “a rich understanding of our social and physical environment,” the authors wrote. Researchers already know that our ability to perceive odors is achieved through olfactory receptors (OR) encoded by 855 olfactory receptor genes. However, about half of these genes in humans are thought to lack function, and are categorized as pseudogenes, leaving a relatively small repertoire of about 400 active olfactory receptor genes.

The reason for the loss of so many of these olfactory genes in humans has remained something of a mystery. “Although functional OR repertoires differ substantially between species, the inactivation of OR genes is particularly massive in the primate lineage for unknown reasons,” the team noted. “OR repertoires have shrunk during human evolution, as suggested by more loss-of-function variants in OR genes than any other gene class and unusually high sequence diversity in intact OR genes compared with other protein-coding genes.” What is also not well understood is how variation in the remaining active genes might influence differences in individuals’ senses of smell. “How does genetic sequence diversity in this unusual class of genes translate to perception and behavior?” the investigators asked.

Scent pens used in the smell test. [Jón Gústafsson, deCODE Genetics – Amgen Inc] To explore this further, Gisladottir, together with co-corresponding deCODE author Kari Stefansson, and team, enlisted 9,122 Icelanders in a GWAS, in search of OR gene variants that influence odor perception. They asked the study participants to smell odors presented to them in pen-like devices that released a particular scent when uncapped. After the participant sniffed each odor pen, the researchers asked them to name the smell. Participants also rated the intensity and pleasantness of each smell. Odors tested included key ingredients found in licorice, cinnamon, fish, lemon, peppermint, and banana.

Through their analysis of genetic loci of interest, the team identified variants in that were linked with differences in the perception and naming of odors. They subsequently confirmed the findings in a separate cohort of 2,204 Icelanders. One of the variants implicated in odor perception was identified in a non-canonical olfactory receptor gene called trace amine-associated receptor 5 (TAAR5). The TAAR5 variant was found to affect the perception of a fish odor containing trimethylamine, a bacterial metabolite that is found in rotten and fermented fish, as well as in other animal odors and various bodily secretions.

In the smell tests, people with a particular variant of the TAAR5 gene were more likely either not to smell anything when presented with the fish odor, or to use descriptors for the smell that were neutral or positive, and not seafood related, such as “potatoes,” “caramel,” and “rose.” The findings are the first to show an important role for this gene in humans, the researchers said. “TAAR5 encodes a member of the TAAR family of trace amine-associated receptors, which are G protein-coupled receptors (GPCRs) that function as chemosensory receptors in the olfactory epithelium of vertebrates, detecting volatile and often aversive amines … To our knowledge, there are no prior reports of a sequence variant in TAAR5 that influences olfactory perception in humans.”

Gisladottir commented, “Carriers of the variant find the fish odor less intense, less unpleasant, and are less likely to name it accurately. There is a lot of animal research on TAAR5 in relation to its role in hard-wired aversive responses to trimethylamine. Our findings extend the implications of this research to human odor perception and behavior.”

A participant in the smell test. [Jón Gústafsson, deCODE Genetics – Amgen Inc] The other two discoveries related to variants in more typical and common olfactory genes. These variants influenced an individual’s ability to name licorice and cinnamon odors. They also influenced the intensity and pleasantness associated with those odors. “We discovered a common variant in a cluster of olfactory receptors which is associated with increased sensitivity to trans-anethole, found in black licorice products but also in spices and plants such as anise seed, star anise, and fennel,” Gisladottir continued.

“Carriers of the variant find the licorice odor more intense, more pleasant, and can name it more accurately. Interestingly, the variant is much more common in East Asia than in Europe.” The cinnamon variant influenced the perception of trans-cinnamaldehyde, the major ingredient in both Chinese and Ceylon cinnamon. Carriers of this variant were able to name the cinnamon odor more accurately, the team reported. Carriers also found the odor more intense.

The combined findings indicate that variation in olfactory genes influences odor perception in humans. “Altogether, our results provide a unique window into the effects of sequence diversity on human olfaction,” the team concluded. “An individual’s personalized OR repertoire gives rise to myriad differences in perception and behavior, including olfactory language, which we are only beginning to understand.”

The newly reported results also show that, while humans have fewer olfactory genes compared to other species, some of the genetic variation that people do carry makes them more, rather than less, sensitive to particular smells such as licorice or cinnamon. “When coupled with evidence for geographical differences in allele frequencies, this raises the possibility that the portion of the extensive sequence diversity found in human olfactory receptor genes that affects our sense of smell is still being honed by natural selection,” the researchers suggested. They aim to continue to collect data on odor perception in people, and also plan to use the same olfactory tasks to investigate smell deficits in the context of COVID-19.


Acknowledgements

This work was supported by R01 DC005782, R01 DC012095, R03 DC011373, R01 DC013339, T32 DC000014 and a National Research Service Award postdoctoral fellowship F32 DC008932 to J.D.M. A portion of the work was performed using the Monell Chemosensory Receptor Signaling Core and Genotyping and DNA/RNA Analysis Core, which are supported, in part, by funding from the US National Institutes of Health NIDCD Core Grant P30 DC011735. A portion of the work was supported by the Defense Advanced Research Project Agency RealNose Project. Collection of psychophysical data was supported by grant # UL1 TR000043 from the Clinical and Translational Science Award program at the National Center for Advancing Translational Sciences. The FACS analysis was performed using the Duke Cancer Institute Flow Cytometry Core. We thank D. Marchuk for sharing equipment, L.B. Vosshall for supervising the collection of psychophysical data and DNA samples by A.K. in her laboratory, and R. Molday (University of British Columbia Centre for Macular Research) for 4D2 anti-rhodopsin antibody.


Methods

Database Searches. Initial tblastn searches for human OR genes were performed using human genome sequences contained in the National Center for Biotechnology Information (NCBI) finished (nr) and draft (htgs) databases (build 32 data) (www.ncbi.nlm.nih.gov/BLAST). Conserved OR sequence motifs used as queries included MAYDRYVAIC transmembrane domain 3 (TM3) and its variants, MALDRYVAIC and MAFDRYVAIC, and KAFSTCASH (TM6). Seven diverse mouse ORs were also used as queries in separate tblastn searches. The short peptide sequences were used in tblastn searches until no new OR sequences were obtained. A nucleotide sequence (≈2 kb) containing each match was retrieved from the database and then translated using ORF Finder (www.ncbi.nlm.nih.gov/gorf/gorf.html) to obtain the encoded protein sequence.

A protein was considered an OR if it was encoded by a coding region of ≈1 kb and contained four OR sequence motifs (GN MAYDRYVAIC, KAFSTCASH, and PMLNPFIY) or their variants at appropriate positions. When sequences satisfying these criteria were used as queries in blastp searches of the NCBI nr database, they invariably showed best matches to known ORs. Sequences with one or more, but not all four motifs, were used as queries in such searches and were considered to be ORs if their best matches were to known ORs. Coding sequences that contained stop codons or frameshifts were counted as pseudogenes, but extremely pseudogenized sequences and isolated gene fragments were excluded.

Sequence Alignments. Nucleotide and amino acid sequences were aligned by using clustalw 1.83 (European Molecular Biology Laboratory–European Bioinformatics Institute, Cambridge, U.K.). The alignments were visually inspected and edited as necessary. The final alignment was used to generate unrooted phylogenetic trees ( clustalw 1.83). Nucleotide and protein sequence identities were determined using the distances function of the Genetics Computer Group (GCG Wisconsin Package, Accelrys, San Diego). The uncorrected distance matrix was then used to assign ORs to subfamilies in which all members of a subfamily were at least 60% identical to all other members in protein sequence. Members of the same subfamily displayed strong phylogenetic grouping. Bootstrap values were generally ≥50%.

To identify the closest human homologs of rodent ORs with known odor ligands (9–14, 16–18), the sequence of each rodent OR was used as a query to search ( tblastn ) the NCBI human genome sequence database (build 32).

The unrooted phylogenetic tree in Fig. 2 was prepared using the protein sequences of the 339 human ORs, the 23 rodent ORs with known odor ligands, and 28 ORs from several species of fish (NCBI).

Phylogenetic tree of sequence relationships among ORs. This tree compares the 339 members of the human OR family, 23 rodent ORs of known function, and 28 fish ORs. Green branches represent fish ORs, and red branches represent human and rodent ORs with known odorant specificities. Odorants detected are indicated near the tip of each red branch. The majority of human homologs of rodent ORs for aliphatic odorants are located in one distinct branch of the tree. This branch (shaded in gray) also contains all of the fish ORs, suggesting a distant evolutionary relationship between receptors for aliphatic odorants and fish ORs.

Chromosome Localization. To determine the physical locations of all 636 human OR genes, the coding region sequence of each gene was used to search ( blastn ) the assembled NCBI human genome database. Using the NCBI map viewer program (www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search), it was possible to determine the chromosomal locations of 630 of the 636 human OR genes.


Methods

Data acquisition and filtering

Natural sequence variations from functionally annotated human ORs [62, 66] were obtained from the Genome Aggregation Database (gnomAD v2, http://gnomad.broadinstitute.org/) using Python (v.3.7.6) data mining scripts. Variant tables for each OR were imported to R (v.3.6.2), including information of chromosome location, transcript consequence, and allele frequencies in seven sub-continental populations (Additionalਏileਁ: Table S1) [21]. Basic Local Alignment Search Tool (BLAST, v.2.10.0) and Python scripts were used to compare the collected sequence information with UniProt database (release 2019_11, https://www.uniprot.org/). The collected data was then filtered to remove null values, duplicates, missing rsIDs, and sequence conflicts with reference Swiss-Prot entries, resulting in a curated dataset of 119,069 nucleotide variants from 378 human OR genes (Additionalਏileਁ: Tables S2-S3).

Topological mapping and BW annotation

Python data mining scripts were used to assign each coding-sequence mutation a topological location according to a structure-based multiple sequence alignment (MSA) of 378 ORs Swiss-Prot reference sequences and class A GPCRs of known three-dimensional structure (Additionalਏileਃ). Natural variants at the TM regions were further annotated with the generic two number system developed by BW consisting of two digits: the first (1 through 7) corresponds to the helix in which the change is located, and the second indicates its position relative to the most conserved residue in the helix (arbitrarily assigned to 50) [24]. This nomenclature was also applied to a 10 residue stretch located between two highly conserved cysteines at the ECL2 (indicated by 45 as the first number attending to its location between the TMs 4 and 5) (Additionalਏileਂ: Figure S7) [25].

Impact evaluation of coding sequence variants

The impact of non-synonymous changes was estimated from the amino acid substitution scores derived from the GPCRtm matrix (Additionalਏileਂ: Figure S3) [33]. In addition, two subsets of BW topological sites were outlined: (i) a functional core (FC) subset of 30 topological positions with a high degree of conservation and likely involved in the receptor activation, G protein binding, or disulfide bond formation (Additionalਏileਁ: Table S4, Additionalਏileਂ: Figure S4) and (ii) a binding cavity (BC) subset of 30 amino acid positions within a distance of ≤𠂔.0 Å to bound ligands in 39 reference class A GPCR 3D structures (Additionalਏileਁ: Table S5, Additionalਏileਂ: Figure S5). This selection exhibited a high degree of correspondence with positions identified in a reference study conducted on orthosteric and allosteric GPCR ligand interactions sites [32], including the 45.52 at ECL2.


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