Personal receptor repertoires: olfaction as a model

Background: Information on nucleotide diversity along completely sequenced human genomes has increased tremendously over the last few years. This makes it possible to reassess the diversity status of distinct receptor proteins in different human individuals. To this end, we focused on the complete inventory of human olfactory receptor coding regions as a model for personal receptor repertoires. Results: By performing data-mining from public and private sources we scored genetic variations in 413 intact OR loci, for which one or more individuals had an intact open reading frame. Using 1000 Genomes Project haplotypes, we identified a total of 4069 full-length polypeptide variants encoded by these OR loci, average of similar to 10 per locus, constituting a lower limit for the effective human OR repertoire. Each individual is found to harbor as many as 600 OR allelic variants, similar to 50% higher than the locus count. Because OR neuronal expression is allelically excluded, this has direct effect on smell perception diversity of the species. We further identified 244 OR segregating pseudogenes (SPGs), loci showing both intact and pseudogene forms in the population, twenty-six of which are annotatively "resurrected" from a pseudogene status in the reference genome. Using a custom SNP microarray we validated 150 SPGs in a cohort of 468 individuals, with every individual genome averaging 36 disrupted sequence variations, 15 in homozygote form. Finally, we generated a multi-source compendium of 63 OR loci harboring deletion Copy Number Variations (CNVs). Our combined data suggest that 271 of the 413 intact OR loci (66%) are affected by nonfunctional SNPs/indels and/or CNVs. Conclusions: These results portray a case of unusually high genetic diversity, and suggest that individual humans have a highly personalized inventory of functional olfactory receptors, a conclusion that might apply to other receptor multigene families.

Relating CNVs to transcriptome data at fine resolution: Assessment of the effect of variant size, type, and overlap with functional regions

Sebastian Martin Waszak

Copy-number variants (CNVs) form an abundant class of genetic variation with a presumed widespread impact on individual traits. While recent advances, such as the population-scale sequencing of human genomes, facilitated the fine-scale mapping of CNVs, the phenotypic impact of most of these CNVs remains unclear. By relating copy-number genotypes to transcriptome sequencing data, we have evaluated the impact of CNVs, mapped at fine scale, on gene expression. Based on data from 129 individuals with ancestry from two populations, we identified CNVs associated with the expression of 110 genes, with 13% of the associations involving complex, multiallelic CNVs. Categorization of CNVs according to variant type, size, and gene overlap enabled us to examine the impact of different CNV classes on expression variation. While many small (

2011

Functional characterization of a human odorant receptor

Valérie Jacquier

Olfaction appears to be mediated by a large family of odorant receptors (ORs) that accounts for ~ 1% of the human genome [1–4]. The functional characterization of these receptors has so far been hindered by their poor functional expression in the plasma membrane of heterologous cells. In order to investigate the problem of inefficient OR membrane targeting, sequential stages in the life cycle of the human OR17-40 were monitored. The modification of the receptor by different tags enabled the direct visualization of its biogenesis and trafficking in HEK293 cells. A multi-color labelling approach, which involved fluorescent subcellular markers as well as the spectral separation of membrane-inserted and intracellularly located receptors, indicated that high overexpression led to the accumulation of the receptor in the ER, whereas low expression levels improved its membrane targeting. To increase the efficiency of OR-mediated signalling, fluorescence-activated cell sorting of a functional OR17-40 – GFP fusion protein was used to separate low from high expressing cells, thus increasing the fraction of odorant-responsive cells up to 80% of the total cell population. Selectively labelled cell surface receptors accumulated over time in intracellular compartments, indicating constitutive receptor internalization. This process, which was independent of receptor activation, occurred along the clathrin-mediated pathway. Moreover, the imaging of single receptors in the plasma membrane indicated that, in absence of ligand, part of the receptors are already confined within small domains of 195 ± 10 nm. This fraction, which increased upon agonist or antagonist binding, probably corresponds to receptors located in clathrin-coated prepits. Odorant molecules structurally related to the cognate agonist helional were tested for their ability to activate OR17-40. It was found that an aldehyde group connected to an aromatic ring via a carbon chain of defined length and containing a methyl group in alpha or beta position is necessary for activating the receptor. In addition, first evidence for an OR17-40-specific antagonism was provided. Finally, OR17-40 activation was monitored in cell-derived native vesicles, opening new ways for assay miniaturization and the development of odorant screenings in a micro-array format.

EPFL2005

Systematic Inference of Copy-Number Genotypes from Personal Genome Sequencing Data Reveals Extensive Olfactory Receptor Gene Content Diversity

Sebastian Martin Waszak

Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95–99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ~15% and ~20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing.

Public Library of Science2010

Functional characterization of a human odorant receptor

Valérie Jacquier

EPFL2005

Systematic Inference of Copy-Number Genotypes from Personal Genome Sequencing Data Reveals Extensive Olfactory Receptor Gene Content Diversity

Sebastian Martin Waszak

Public Library of Science2010