Who, me? I don't believe in single-gene causation! (or do I?). Part I. What does it mean?

We were told in no uncertain terms the other day that no one believes in single gene causation anymore.  Genetic determinism is passé, and everyone knows that most traits are complex, caused by multiple genes, gene x environment interaction, or if they're really sophisticated, epigenetics or the microbiome. But is this the view that investigators actually follow?  That's not so clear.

We all throw around the word 'complex' as if we actually believe it and perhaps even understand it.  Of course, (nearly) everyone recognizes that some traits are 'complex' meaning that one can't find a single clear-cut deterministic cause, the way being hit on the side of the head with a baseball bat by itself can send one out for the count.  But in fact the hunt for the gene (or, alright if you insist!) genes 'for' a trait is still on. You name your trait: cancer, diabetes, IQ, ability to dunk a basketball, or get into Harvard without a Kaplan course, and someone's still looking for the gene that causes it.

It is this push to find genes for traits, despite all sorts of denials, that fuels the GWAS and similar fires.  Caveats notwithstanding (and usually offered just to provide technical escape lest one is wrong), that is what the promised 'personalized genomic medicine' in its various forms and guises is all about.

So let's take a careful, and hopefully even thoughtful look at the idea of genetic causation.  We are personally (it must be obvious!) quite skeptical of what we think are excessive claims of genetic determinism (or, now, microbiomial determinism), but they are still being made, so let's tease a few of them apart.

Single gene causation does exist, at least sometimes (doesn't it?)!
First, though, what do we mean by the word 'causation'?  Generally, we think people mean that gene X or risk factor Y is sufficient to cause trait Z.  But, it might also mean that gene X or risk factor Y are necessary but not sufficient causes of trait Z.  The baseball bat might have caused your concussion, but in fact someone had to swing it.

There are well-documented single risk factors, genetic and otherwise, that everyone accepts 'cause' some disease in a very meaningful sense. Examples are some alleles (variant states) of the CFTR gene and Cystic Fibrosis (CF), BRCA1 and 2 variants and breast cancer, or smoking and lung cancer.  Having the alleles associated with CF or breast cancer, or being a long time smoker do put people at high risk of disease.

But, for these and other examples there are usually healthy people walking around with serious mutations in the gene, or heavy smokers who enjoyed their cigarettes well into old-age.  Gene X or factor Y aren't sufficient to cause trait Z.  There are also hundreds of genetic variants found in patients that are assumed to be causal, but for elusive reasons (for example, mutations in non-coding regions near to the CFTR coding regions that have no known function).  What is it about gene X that causes trait Z?  We don't know, but gene X looks damaged in this person, so it must be causal.

The CF case is interesting.  This is an ion channel disease.  Ion channels are gated openings on the cell surface that pass sodium, potassium, calcium and other ions into and out of the cell in response to local circumstances.  CF is characterized by abnormal passage of chloride and sodium through ion channels, causing thick viscous mucous and secretions, primarily in the lungs but with involvement of the pancreas as well.  If the ion channel is badly built, or doesn't get to the surface of cells lining various organs like pancreas and lungs, then the cell cannot control its water content, secretion, or absorption, and the person with the malfunctioning channels has CF.  Again, gene X causes trait Z.

But there are gradations in channel malfunction, and gradations in severity of the disease, and we have no way to know how many people are walking around with variants but no actual disease.  Here, we can say that when it happens, CFTR mutations do cause the trait in the usual way.  But what about when there are mutations but no disease?  Gene X doesn't cause the disease after all?  Or disease and none of the known causal mutations?  Wait, we thought gene X caused the disease?  Could we be assuming single gene causation, and looking only at the CFTR gene, rather than at many other aspects of the genome that may affect ion channels in the same cells or, indeed, may cause the trait in a way we could understand if we but identified them?  This is an open question--but it applies to many other purportedly single-gene diseases.  Gene X and some other gene/s, or some environmental factor cause the disease in at least some instances.  Is it simple or isn't it?

The BRCA story is also interesting.  A BRCA1 variant associated with disease does not lead directly to cancer.  Instead, BRCA1 is a gene that detects and repairs genomic mutations in breast (and other) cells.  If you have a dysfunctional BRCA1 genotype, you are at risk of some one breast cell acquiring a set of mutations that don't get detected and repaired.  What causes those mutations?  Some happen when cells divide, so the activity of breast cells affects the rate of mutational accumulation.  Other lifestyle factors do as well (parity, age of childbearing, lactation and apparently things like diet and exercise). And a person with a causal BRCA mutation lives perfectly healthfully for decades, which if you think in classical Mendelian terms, would not happen if s/he had a 'bad' gene.  BRCA doesn't exactly cause cancer, but it allows it to be caused.  Gene X plus time plus environmental risk factors cause the disease.  Though, we all believe it's a single gene, BRCA1 or 2, that causes cancer.

The obvious non-genetic instance, smoking and lung cancer, is similar but not exactly the same.  Smoking is, among other things, a mutagen: it damages genes.  So one, if not the major, reason for the association is that the mutations caused by smoke can damage genes in lung cells that lead those cells to proliferate out of control.  The reason the risk is probabilistic -- that is, a smoker doesn't have a 100% chance of getting lung cancer -- is that it's impossible to know how many or which mutations a given person's smoking has led to.  In fact, smoking is only an indirect cause, since it is mutant genes in lung cells that, after accumulating in an unlucky way, start the tumor.  Still, in this case, knowing how much a person has smoked can allow one to estimate in some probabilistic way the relative risk of lung cancer due to enough mutations having arisen in at least one lung cell. Still, many who smoke don't get cancer, and many get cancer who don't smoke.  Since smoking, and a few other such risk factors (e.g., exposure to asbestos, and some toxic chemicals) have strong effects,  even if probabilistic, everyone is generally comfortable with thinking of them as causal.  Risk factor Y causes trait Z.  But, in fact, risk factor Y plus time plus unlucky mutations cause trait Z.

Deterministic genotype 'pseudo-single-gene' causation?
More problematic are 'complex' traits, that clearly are not due to simple single gene variants--they don't follow patterns of trait appearance in families that would be consistent with simple Mendelian inheritance.  The number of such traits is legion, and is driving the GWAS industry as we have many times commented on.  They are typically common in the population.  They are complex because we feel--know, really--that many different factors combine somehow to generate the risk.  Most instances are not due to one factor alone, though some may be in the sense of BRCA and CFTR.  So, we do something like genomewide association studies to try to identify all the potentially causal parts of the genome (and, similarly, but less definitively as a rule, lifestyle factors, too).  Here, we'll assume that all of the genomic variants that might contribute are known and can be typed in every person (this is, as everyone knows, far, far from being true at present, if it's even possible).

Advocates can deny any element of single-gene thinking in GWAS reports, where hundreds of loci are claimed to have been found, but these are treated as causal, and major journals are filled to the gills with papers with titles to the effect of "five novel genes for xyz-itis".  This is the slippery slope of simple causation thinking.

If multiple factors contribute, what we know is that most do so only probabilistically in the above senses.  That there are other things at work is rather obvious for the many traits that even in those at high risk don't arise until later or even late in life.  And, in reality, it is nearly always true that cases of the disease are associated with individually unique combinations of the risk factors.  So, your personalized risk is computed as some kind of combination, like the sum, of your estimated risk at each of the putative sites, R= R1+R2+R3...., where at each site one allele is given the minimal risk (or, perhaps, zero) and the other allele the risk estimate by the difference in prevalence of the trait in cases vs in controls.  This might be considered the very opposite of single-gene causation, but conceptually it's pretty much the same, because it treats your aggregate as a single kind of risk score, as if it were acting as a unit.  The idea would be completely analogous to the risk associated with a specific variant at the CFTR or BRCA gene.  Your genotype as a whole would be viewed essentially as a single cause.

These are examples of causal rhetoric.  But these causes are probabilistic.  What does that mean?  It means that you are not 100% protected from getting the trait, nor 100% doomed.  Your fortune is estimated by some number in between.  We call it a probability, estimated either from the presence of one risk-factor or from the fixed set that you inherited.  But what does that probability mean, and how do we arrive at the value and how reliable is it?  Indeed, how often can we not even know how reliable it is?

These are topics for tomorrow.

The complexity of simple genetic disease

Cystic fibrosis is an ion channel disease that interrupts the flow of salts and fluids into and out of cells, and this affects multiple organs. The most serious consequence of the disease is the production of thick mucus in the intestines and lungs, which leads to respiratory complications, the leading cause of death among people with CF.

Cystic fibrosis is an inherited disease.  The causative gene, CFTR, was identified 22 years ago.  Over 1000 mutations associated with CF have been identified since then, many seen in only one patient or a single family.  In the US the most common mutation is F508del; this designation means that the amino acid that is normally the 508th amino acid along the chain that makes the CFTR protein has been deleted. Another mutation, G551D, is found in about 4% of patients in the US -- this mutation replaces one amino acid with another at the 551th position in the protein chain.  

Identifying the CFTR gene created quite a lot of excitement about the potential for gene therapy, but the initial enthusiasm was pretty quickly dampened by the difficulty in transporting a normal copy of the gene to the required sites in the body. A different therapeutic approach was described in a paper in PNAS in 2009.

Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (P_o) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl⁻ secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na⁺ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway. 

The pharmaceutical company that makes VX-770 has just announced the successful completion of a 48 week clinical trial of the drug. The results are impressive. Lung function was significantly improved, and

[h]ighly statistically significant improvements in key secondary endpoints in this study were also reported through week 48. Compared to those treated with placebo, people who received VX-770 were 55 percent less likely to experience a pulmonary exacerbation (periods of worsening in signs and symptoms of the disease requiring treatment with antibiotics) and, on average, gained nearly seven pounds (3.1 kilograms) through 48 weeks. There was a significant reduction in the amount of salt in the sweat (sweat chloride) among people treated with VX-770 in this study. Increased sweat chloride is a diagnostic hallmark of CF. Sweat chloride is a marker of CFTR protein dysfunction, which is the underlying molecular mechanism responsible for CF. People who received VX-770 also reported having fewer respiratory symptoms.     

This is exciting news for the CF community, even for those who don't have the G551D mutation, because the same company is currently testing a drug to correct for the effects of the F508del mutation.

In people with the G551D mutation, CFTR proteins are present on the cell surface but do not function normally. VX-770, known as a potentiator, aims to increase the function of defective CFTR proteins by increasing the gating activity, or ability to transport ions across the cell membrane, of CFTR once it reaches the cell surface. In people with the F508del mutation, CFTR proteins do not reach the cell surface in normal amounts. VX-809, known as a CFTR corrector, aims to increase CFTR function by increasing the amount of CFTR at the cell surface. 

This all has the potential to change the future for people with CF.  And it also means that if the function of a gene and mutations in that gene are understood, the parameters are there for potentially developing therapies.  We seem to understand a lot about this ion channel.  In fact, if these results are real--general, long-lasting, and clinically or lifestyle-important as well as statistically significant -- they probably will apply to many other CF patients with other mutations that are individually rarer but have similar effects on the CFTR protein. 

But, the gene for CF has been known for 2 decades, and a treatment for just  4% of people with the disease is only now beginning to look promising.  The difficulty of getting to just this point is a sobering reminder that 'personalized medicine' is going to be an order of magnitude harder for polygenic diseases.  And hopefully, we won't have to take back these positive feelings about these potentially life changing results, and this 48 week trial is not being reported prematurely to boost stock prices or anything cynical like that.

If it works as the current story suggests, these results exemplify what we personally have repeatedly said about medical genetics.  There really are good ways to spend genetics research effort, not on mindless GWAS mapping, but on traits that are tractably simple and that really are genetic in a meaningful sense.  This seems like a very good example of that principle even if, as is the case with other instances, only a fraction of all CF patients will benefit directly.

Genetics in clinical practice--the tail end of the spectrum?

One of the more interesting talks at the Bristol meeting, perhaps because it's an area we don't know much about, had to do with clinical applications of genetics. The question concerned when, where, and how genetic information can be useful in the clinic. But the issues go much deeper than that, and relate to main points in our 'Mermaid' book.

The idea presented was that genetic information can be useful, not so much for disease risk prediction, because genotypes have poor predictive power for 'complex' traits (that is, and somewhat circularly, those that are not due to a single, and hence predictive, gene!). Instead, the argument was that genotyping can help determine therapy in the diagnostic sense of determining which of many causes may apply to, and hence guide therapy for, individual cases of a given disease.

An example given was MODY, or 'maturity onset diabetes of the young. MODY runs in families and can mimic both type 1 and type 2 diabetes, though most often it is a mild form of T1D, but with patients continuing to make some insulin (T1D, or juvenile diabetes is due to a failure to make insulin; T2D, or adult diabetes, is failure of cells to respond to insulin). The speaker, Dr Andrew Hattersley, described one family in which each affected member was being treated differently, but once the causal gene was known, each member was treated more appropriately, and the disease then better controlled. This was very good to hear because all too often even knowing the causal gene can't inform treatment--Huntingdon's disease comes to mind.

Hattersley also discussed a gene that causes three forms of achondroplasia, or dwarfism, but the particular mutation in that gene determines whether an individual will be somewhat shorter than normal, or will be much more severely affected, and knowing the mutation during pregnancy helps to prepare the family and clinician.

He primarily discussed single gene disorders, although these are often disorders that can be caused by a number of different mutations or even different genes, but in the instances described, knowing the specific mutation can make the difference between useful medical intervention and none. The idea is essentially that the 'same' disease is not really the same in different individuals if enough specificity is known, and that when a gene's effects are understood, knowing the causal gene is clinically relevant.

By contrast, all too often in human genetics identifying genes whose variation is statistically associated in at least some studies with the occurrence of disease contributes nothing toward treatment, because the statistical connection is too weak to be useful. That is the problem that is now widely recognized in regard to GWAS (see our many earlier posts) for complex diseases, so we were heartened to learn that this is not always the case. In some cases, knowing the physiological pathway is very useful.

The much broader relevance of these points is that they relate to genetic determinism: the degree and specificity to which genes determine phenotypes (the same applies to environmental factors). Only to the extent that genes determine traits in organisms can knowing the genotype be used to predict the trait. That has everything to do with views, accurate or hyped, about the usefulness of huge genotyping studies to health. It has to do with the use of genetics in social behavioral and other societal contexts. And it has to do with the origins of traits and with evolution itself.

To the degree that natural selection molds and creates (produces) the traits we see in organisms--and that it does so is at the heart of Darwinian theory--genes must determine traits. That's because if genotypes do not determine the trait, selection for or against variation in the trait won't affect the frequency of genetic variation, and hence won't guide genetic evolution. Yet the modern Darwinian evolutionary theory is entirely based on genetic determinism.

With selection on phenotypes, not genotypes, if the connection between genotype and phenotype is weak, selection only weakly affects the relative success ('fitness') of genotypes, and their frequency changes over the generations will largely be the result of chance, not selection.

When a mutation strongly affects a trait, and the trait is important to organisms' success, then Darwinian theory works just fine as advertized. But this is not so if traits are so genotypically complex that selection hardly affects an individual gene.

There is a middle ground. If a trait, like a disease, is a strong reflection of a specific genotype, but many different genotypes can generate similar traits, then prediction can be weak but once the trait has arisen it can be useful to determine which of those causes is responsible. From an evolutionary point of view, if each instance of a trait is due only to variation in a single gene, then selection can affect the frequency of that variation.

The key difference is that for complex traits the phenotype is thought to be the simultaneous result of variation at many different genes. There are so many combinations of genotypes that can generate similar phenotypes, that one can't usefully predict the latter from the former--or, perhaps, not even go back the other way, unless variation at only one or a few genes is responsible in a given instance.

Thus, prediction in development, medicine, or evolution all depend centrally on the degree to which genes individually, or in aggregate, determine the traits you bear in life. In the 'tail' end of the causal spectrum, where specific variants exert strong effects, things work out nicely.