Genetic elucidation of human hyperosmia to isovaleric acid

Abstract

The genetic basis of odorant-specific variations in human olfactory thresholds, and in particular of enhanced odorant sensitivity (hyperosmia), remains largely unknown. Olfactory receptor (OR) segregating pseudogenes, displaying both functional and nonfunctional alleles in humans, are excellent candidates to underlie these differences in olfactory sensitivity. To explore this hypothesis, we examined the association between olfactory detection threshold phenotypes of four odorants and segregating pseudogene genotypes of 43 ORs genome-wide. A strong association signal was observed between the single nucleotide polymorphism variants in OR11H7P and sensitivity to the odorant isovaleric acid. This association was largely due to the low frequency of homozygous pseudogenized genotype in individuals with specific hyperosmia to this odorant, implying a possible functional role of OR11H7P in isovaleric acid detection. This predicted receptor-ligand functional relationship was further verified using the Xenopus oocyte expression system, whereby the intact allele of OR11H7P exhibited a response to isovaleric acid. Notably, we also uncovered another mechanism affecting general olfactory acuity that manifested as a significant inter-odorant threshold concordance, resulting in an overrepresentation of individuals who were hyperosmic to several odorants. An involvement of polymorphisms in other downstream transduction genes is one possible explanation for this observation. Thus, human hyperosmia to isovaleric acid is a complex trait, contributed to by both receptor and other mechanisms in the olfactory signaling pathway.

Figure 1. Olfactory Threshold Distributions

Histograms of the measured olfactory thresholds for four odorants: IAA (A), IVA (B), LCA (C), and CIN (D). Odorants are expressed in molar (M) concentrations (in the oil solution) and were tested in 377 genotyped individuals, except CIN, which was tested in a randomly selected subsample of 200 participants. The data are presented for both genders pooled together. A threshold score of one indicates individuals who could not detect the highest possible odorant concentration (10⁻² M). Red, yellow, and green, respectively, represent fractions out of the total sample for homozygote disrupted, heterozygote, and homozygote intact OR11H7P genotypes.

Figure 2. Genotype–Phenotype Association

ANOVA p-values for comparison between the olfactory threshold distributions of participants with homozygous disrupted genotypes versus heterozygous and homozygous intact genotypes (1 df), using “gender” and “gender by genotype” as covariates. SPG loci are enumerated as in Table S2. The broken line indicates the statistically significant value of p = 0.05 after Bonferroni correction for 172 tests (four odorants × 43 SPGs). The two strongest p-values for IVA are for the genes marked in Figure 3. Using the individual's average threshold towards the four odorants as an additional covariate did not change the association signal with OR11H7P (open circle in IVA panel). The association signal with OR4Q2P is reduced after adjusting for the OR11H7P effect (solid square in IVA panel).

Figure 3. The Genomic Region Associated with IVA Hyperosmia

OR genes are represented by red, green, and yellow triangles indicating pseudogenes, intact genes, and SPGs, respectively. The two SPGs showing the strongest association with IVA sensitivity are marked with single and double asterisks. A linkage disequilibrium plot of SNPs with minor allele frequency ≥ 5% in HapMap CEU [46] is depicted for this genomic region. The pairwise r ² values are indicated on a gray scale, with black = 1 and white = 0. This region contains also the putative OR-specific trans-acting enhancer element (H) [47,48].

Figure 4. OR Responses to IVA

(A) Representative current traces from Xenopus oocytes expressing the OR11H7Pi, OR52E4, OR11H4, or OR11H6 receptor (see Materials and Methods) challenged with 15-s applications of 3 mM IVA and 1 mM IBMX. (B) Summary of the 4–12 recordings from oocytes expressing OR11H7Pi, OR52E4, OR8A1, OR12D2, OR11H7P, OR11H4, or OR11H6. Responses were normalized to the 1 mM IBMX response in the same oocyte and are presented as mean ± standard error of the mean. Significant differences from OR52E4, OR8A1, and OR12D2: *, p < 0.05; **, p < 0.01. Significant differences from OR11H7P: ^†, p < 0.05; ^††, p < 0.01.

Figure 5. Excess of General Hyperosmia

(A) Histogram of average olfactory thresholds (adjusted for gender). The average threshold values for the four odorants were calculated for the raw data (black bars) and for data generated by 1,000 permutations of the individual odorant thresholds (dotted bars). The significantly broader distribution of the original data as compared to the permutated data (ANOVA, F = 1.83, p = 1.68 × 10⁻¹⁰) indicates an excess of individuals with extreme threshold values, particularly in the hypersensitivity end of the distribution. (B) Combinations of odorant thresholds (adjusted for gender) for the four odorants. Shown are hyperosmia (lowest 10% of thresholds in the entire sample, black), normosmia (middle 80%, gray), and hyposmia (highest 10%, white). Individuals with similar threshold patterns are clustered together. For clarity, only 50 of the total of 123 normosmic individuals are shown. The probability of observing three individuals defined as generally hyperosmic (i.e., having hyperosmia to all four odorants) in this cohort is computed as ∼10⁻¹².