Ectopic thinking?

Our gene mapping project, which we first blogged about here, is starting to get interesting.  And not necessarily for the reasons that we'd hoped.  You might remember that our project is looking for genes involved in variation in specific craniofacial traits in the F34 generation of descendants of a cross between two inbred mouse lines.  We've measured a handful of traits in over a thousand mouse skulls, and mapped their genetic effects by looking for genetic variation across the genome that might be associated with variation in the traits.

Sparing you the gory details, we'll just say that the chromosomal intervals that one or more of the traits mapped to span 30% of the genome, and 10% of all coding genes. That's 2400 genes or so that could potentially be of interest in affecting head shape in just these particular mice, with the restricted genetic variation they have (because they are descendants of only two inbred parental mouse strains).  That's a lot of genes to wade through to figure out which might be most likely to be involved in the traits we're looking at.

One way to prioritize candidate genes from such a study is to look for the genes in every interval that you know from prior work to be involved in your trait of interest.  Or to identify genes in families that include genes involved in your trait of interest -- these would then be considered guilty by association.

But this means most genes don't have a fighting chance of being considered, because you don't happen to know anything about them, or because nobody knows anything about them, or because what's known about them only partially represents what they do. 

To try to minimize this, many people automate the search, with programs that cull the genes that the literature indicates might be of interest, or that seem to be expressed where you want them to be.  So, this might solve the problem of no one knowing everything about all genes, but it doesn't solve the problem of nothing being known about so many genes, or that there's only partial knowledge.  And it doesn't solve the problem of having to tell the program what to look for, which means you're constraining it in the same way you would if you were doing the search by hand, looking for specific families of genes.  Nor, of course, does it solve the problem of what's happening in all the non-coding DNA that flanks all those genes. 

Thus, we decided that the least biased way to comb the data was to go through all the genes in all the intervals by hand.   We're still making sense of all that, not least because we are hoping not to be constrained by the usual ideas about statistical significance, but we've learned some interesting things along the way.

For example, one of the intervals of interest is loaded with olfactory receptor (OR) genes.  Olfactory receptors reside on the cell surface of olfactory receptor neurons, and are involved in odorant detection.  ORs form the largest family of genes in many genomes -- about 1000 different genes -- and they cluster in sets of genes in various locations on a number of chromosomes.  ORs have a distinctive expression pattern, with only one expressed per neuron in the tissue lining the nose, where they each are sensitive to particular aspects of molecules the animal inhales, and hopes to smell.  How expression of the remaining 999 genes in each cell is blocked is still not known.

ORs are an interesting example of something we've blogged about before, but that continually surprises us.  One of the ways we're evaluating the possible role of all these genes in development of the traits we're looking at is to look at where they are expressed in the developing embryo.  We initially thought this would be helpful for narrowing the search, but it turns out that about 95% or even more of genes (for which there are expression data) are expressed in the head (80% alone in the brain), so it's turning out that expression isn't all that helpful for narrowing the search.  But it does mean we've looked at images of gene expression for around 2000 genes.

Olfr66, GenePaint, E14.5

And ORs are a good example of how what we think we know can inhibit our understanding.  Here, e.g., are the expression results for olfactory receptor 66 (Olfr66) in a developing mouse (at embryonic day 14.5).  Just to orient you if you're not used to looking at such images, it's a single front to back section, the snout halfway down the image and pointing to the left, and the tail at the bottom.  The dark blue is a stain showing cells where the gene is expressed at this particular stage of development.  It's no surprise to see it in the olfactory epithelium in the snout, but notice that it's also in the axial skeleton (vertebral column), probably in cartilage cells that will soon become bone.

What's it doing there?  These are olfactory receptors!  You don't smell with your backbone!  In fact, a lot of ORs are known to be expressed outside the olfactory region, particularly in the testes, but also in the spleen, the thyroid, salivary glands, the uterus, the skin, and other tissues.  A 2006 paper is of interest in this regard, not only because it documents non-olfactory related expression, but because of its title -- "Widespread ectopic expression of olfactory receptor genes".  Ectopic expression, meaning expression where it's not supposed to be. 

But it's only not supposed to be expressed in the axial skeleton because that's not where its name says it will be, not because Nature says so!  People named these genes!  And, there is some discussion in the paper about how ORs might be involved in chemotaxis of sperm as they try to reach and penetrate the egg -- how they direct their movement, based on chemicals in their environment.  Which is equivalent to assuming they are essentially carrying out their olfactory function in the testes, where a different form of molecular reaction than odorant-detection is going on.  But, what about in cartilage, in the image above?  It's hard to imagine chemotaxis has anything to do with OR function here.

Well then, maybe it's an experimental artifact -- maybe the experiment picked up expression of a gene sort of like Olfr66, but not quite, along with Olfr66?  Maybe.  But, then we'd have to explain away all the expression studies showing non olfactory expression of many ORs, and it's rather unlikely that it's all due to experimental artifact.  This is how our own assumptions constrain what we know or even want to know about the function of so many genes.  Maybe Olfr66 has a function we don't yet understand.  As do other ORs.  And, by extension, so many other genes. 

But calling unexpected expression 'ectopic', or naming genes based on only a single role, or in their involvement in disease, when they have other perfectly normal functions, are ways of building in assumptions that, once accepted, can keep us from recognizing that there's a lot we don't yet understand about genes.

The genetics of olfaction -- is a rose a rose by any other nose?

Laura Spinney writes about the genetics of olfaction in The Independent this week, and about population variation in what we can smell.  The genetics of olfaction have been pretty well worked out; in fact, the 2004 Nobel Prize in Physiology or Medicine went to Linda Buck and Richard Axel for this work.  But there is much still left to understand. 

Genes for olfactory receptors (ORs) comprise the largest gene family in the mammalian genome -- at about 900 genes and pseudogenes in humans, they make up approximately 3% of our total genome. On average, we've each got about 400 working olfactory receptor genes, and the rest have accumulated enough mutations to no longer be functional, and are now pseudogenes.  This is not quite accurate, because we are diploid so that we actually have about 800 OR genes, 2 copies of the 400.  Of the hundreds of these genes, many are polymorphically pseudogenes, meaning their sequence works in some copies in the population, but has been mutated out of function in others.

In addition, the copies we each have of each of our functional OR are themselves highly variable in their odorant binding pockets (the part of the receptor that binds to odorant molecules that float by in the nose).

"When I give talks, I always say that everybody in this room smells the world with a different set of receptors, and therefore it smells different to everybody," says Andreas Keller, a geneticist working at the Rockefeller University in New York City. He also suspects that every individual has at least one odorant he or she cannot detect at all – one specific anosmia, or olfactory "blind spot", which is inherited along with his or her olfactory apparatus.

Each olfactory receptor responds to several odorants.  But with so many genes, how on earth could we actually distinguish what we're smelling?  The answer is remarkable and one of the most interesting unsolved problems in genetics.  Our hundreds of OR genes are located in many different clusters of a few to hundreds of adjacent duplicate OR genes.  And we have two copies of each.  But in each individual olfactory neuron, only one of these hundreds of genes is expressed!  The other 799 genes, all over the genome, are inactive.

From Genetics and the
Logic of Evolution,
Weiss and Buchanan, 2004

Further, neurons expressing a given OR are in some way guided by that choice to collect in OR-specific locations in the olfactory bulb of the brain.  That's what makes it possible for the brain to tally what specific receptors have been triggered.  That is, the binding of an odorant to its receptor triggers its perception in the brain.  The wiring pattern is to some extent, at least, conserved among individuals, so in that sense we may share not only the ability to identify, say, 'lemon', but to experience it in somewhat similar ways.

Most odor perception involves a combination of signals from a number of different receptors.  And, some of those receptors may be non-functioning pseudogenes in some people, and if the subset of pseudogenes differs, the odor perceived will be different.

That genetic variability is reflected in behavioural variability, as Keller, with colleague Leslie Vosshall and others, recently demonstrated when they asked 500 people to rate 66 odours for intensity and pleasantness. The responses covered the full range from intense to weak, and from pleasant to unpleasant, with most falling in the moderate range – a classic bell curve in each case.

There is also variation in how intensely people can smell.  Some people are born with no ability to smell, while others are acutely aware of odors.  This may have nothing to do with odorant receptors, but instead how efficiently the odorant signal is transmitted to the brain.

One of us (Ken) spent a sabbatical in England working with Manolis Dermitzakis at the Sanger genome institute outside of Cambridge, trying to find genomic DNA sequence 'signals' that might help explain the extensive monoallelic and monogenic expression of only one OR per olfactory neuron.  Unfortunately, the search was frustratingly unfruitful. In fact, earlier we had done a study in the Pennsylvania Amish, collaborating with one of the experts cited in the Spinney story to see if, in this classic founder-effect population, we could work out the genetics of the ability to identify specific odorants, for which the ability to smell seemed, in other studies, to be polymorphic.  We couldn't find any pattern, because essentially everyone could smell all the test odorants.

The unusual expression pattern of this huge gene family, and the bookkeeping by which its detection of complex odorants allows us to identify our surroundings with a great deal of reliability (and, much more so in other species like dogs, who use the same system) is one of the remarkable, and incompletely understood, facts of genetic life.