Tuesday, March 27, 2007

Silent Mutations and Neutral Theory

 
This is a post about the quality of science writing and what can be done about it. I'm picking on an article in SEED magazine here but it's not because SEED is any worse than the competition. It's partly because SEED makes claims about raising the quality of science writing and science education. For example, this statement from a SEED press release seems to indicate that they aspire to better science writing than the competition [Seed Media Group Adds Scientific and Political Pundits to Editorial Team] and certainly their commitment to science bloggers suggests the same aspiration.
As part of its growth strategy, Seed Media Group will develop original science content aimed at a general audience for distribution across a number of media channels, including magazines, books, newspapers, online, topical blogs, digital, film and television. Seed Media Group's endeavors will present science in the same culturally articulate and accessible style that earned Seed a prestigious UTNE Independent Press Award in 2004 and the support of leading advertisers.
It's reasonable, in my opinion, to expect that SEED will live up to this billing. They've certainly made a major step in that direction by hiring PZ Myers to write a monthly science column. The science in his first two contributions is impeccable. As we will see, it raises the question of whether you need to be a scientist in order to get the science right. I hope that's not true.

I'm not going to criticize PZ's articles. Instead, I want to examine another article published in the March 2007 issue of SEED. (That's the one with the "TRUTH" prominently displayed on the cover!) The article in question is titled The Sound of Silence and it's written by Lindsay Bothwick, an experienced science writer with a M.Sc. from McGill and a Masters degree in journalism from Ryerson University here in Toronto. She's a senior editor at SEED so I'm assuming she can take criticism.

The article talks about silent mutations in protein-coding regions. The focus is on a recent Science paper showing that some silent mutations affect the activity of a protein. The point I will make is that the SEED article is very misleading and misrepresents the state of knowledge in this field.

Before getting into the article, let me give you some background.

The most common kinds of mutations are those where one nucleotide is substituted for another. For example, a G or an A or a T replaces a C. This substitution usually results from an error during DNA replication.

If the mutation (allele) persists in a population, it's called a single nucleotide polymorphism or SNP (pronounced snip). The term polymorphism means that there are at least two different alleles segregating in the population. Often these are the original "wild-type" allele and the new mutant allele.

We now recognize that genomes within a population are very heterogeneous. Polymorphism is common. This level of variation was discovered in studies during the 1960's and it's much higher than most scientists thought prior to 1960.

There are three explanations that can, in theory, account for this high level of polymorphism

First, if we think about SNP's, they can represent a transient phase of fixation by natural selection. In this case, one of the alleles is rapidly replacing the other and we just happen to catch it in the act. Back in the days when natural selection was the only game in town it was thought that this transient stage would be rare so populations were not expected to show much variation.

Polymorphism can also be explained by balancing selection. This is when the population has to maintain several different alleles because there is selection for heterogeneity. The classic example is the mutation for sickle cell anemia. When a person is homozygous for the mutant allele they exhibit the symptoms of anemia but when heterozygous they are resistant to malaria. Balancing selection is not common and it can't explain the variation that was discovered in the 1960's.

The third explanation was that the variation is mostly neutral. The idea here is that the majority of mutations are not being acted upon by natural selection. They are not being removed by purifying selection; they are not being maintained by balancing selection; and they are not rising to fixation under positive selection. It was the discovery of significant polymorphism in populations that gave rise to Neutral Theory in the first place ((Kimura, 1968, King and Jukes, 1969).

Neutral mutations will eventually become fixed or be eliminated from the population and the change in frequency is due entirely to random genetic drift. Drift is a much slower process than natural selection so there will always be large numbers of neutral alleles in the process of becoming fixed or extinct.

Neutral Theory and random genetic drift explains variation and it also explains molecular evolution and the (approximate) molecular clock. There are no other explanations that make sense and nobody has offered a competing explanation since Motoo Kimura (1968) or Jack King and Thomas Jukes (1969) published their papers almost fifty years ago. (Aside from occasional nitpicks, of course. There are always scientists who like to show that some mutations that were thought to be neutral are actually beneficial or deleterious. None of them have mounted a serious claim that most variation or most of molecular evolution can be explained by natural selection.)

The history of variation and the competing explanations were well covered by Lewontin in his 1974 book The Genetic Basis of Evolutionary Change. (Lewontin published the classic 1960's papers that revealed extensive within population variation.)

Long-held assumptions about "silent" genetic mutations have been torn down, challenging a fundamental evolutionary theory.

Lindsay Borthwick
SEED
March 2007
This brings me to the article in the March issue of SEED [The Sound of Silence]. It begins with a conclusion that's all too common in popular science writing these days,
Scattered throughout the human genome are thousands of mutations that biologists have treated mostly as footnotes. They're hardly few in number—in coding regions of the genome, there are as many as 15,000—but biologists regard them as mutations that simply don't change the way a cell functions. Both in name and effect, they have been accepted as "silent." Now, however, new discoveries are showing that silent mutations appear to play an important role in dozens of human genetic diseases, a fact that is forcing biologists to discard a long-held evolutionary theory and to reexamine the very rules governing the transfer of information from DNA to proteins.
What's going on here? Has there been some extraordinary new discovery that's about to overthrow evolutionary theory and the "rules" of information flow? I will attempt to show that this rhetoric is completely unjustified. It presents a misleading picture of the state of modern science.

The author is talking about silent mutations. These are mutations in the coding region of a gene that alter a codon without changing the amino acid. The genetic code is redundant because there are 64 possible codons and only 20 amino acids. This means that several amino acids have multiple codons. For example, there are six codons for leucine (Leu): TTA, TTG, CTT, CTC, CTA, and CTG. If an original TTA codon is mutated to CTA then it still specifies leucine and this is a silent mutation.

The concept of codon bias has been known for almost forty years and it's an important part of all university courses in molecular biology. Some codons are more efficient than others during translation because the levels of various tRNAs in a cell are not identical. A rare codon will be translated less efficiently because the tRNA that binds to it will not be recognized as frequently as the codon for more abundant tRNAs. There are published codon usage tables for most species showing the preferred codons in that species. Highly expressed genes will preferentially use the codons recognized by the abundant tRNA species. All students know what these tables mean. It means that not all silent mutations are neutral. (It's on the exam!)

This is only one possible reason for silent mutations not being neutral. The various possibilites were discussed by Jukes back in 1980 when he revisited the evidence for neutral changes (Jukes, 1980)) . He gave some specific examples and then addressed the theoretical problem,
The question arises, are these silent changes actually neutral, or have they taken place for adaptive reasons, such as the requirement for a specific secondary structure in mRNA, or a preferential use for certain transfer RNAs in regulating the rate of synthesis of a protein?
For some results the answer is that the silent changes really are neutral, although in a few cases there is evidence of adaptation. The point is that these issues have been recognized and dealt with for decades.

The existence of a few exceptions to a rule does not invalidate the generality. That's an important point. It's one that all science journalists need to grasp. There are no absolute, inviolate, rules in biology. The generalities are all about relative frequencies. Are most silent mutations neutral or are most subject to natural selection?

As Jukes put it 27 years ago,
The neutral approach to molecular evolution is a proposal to prove a negative, which is something like trying to show that a given substance is not a carcinogen. The counterrresponse to the publications by Kimura (1968) and King & Jukes (1969) has been quite strong. Any exceptions to neutrality are usually taken as disproof of it, and many authors have cited such exceptions for this purpose. We have, indeed, developed evidence for such exceptions ourselves, because a theory should be challenged by those who have postulated it.

For example, the finding that synonymous codons for each amino acid are not use in equal amounts in β-hemoglobin mRNA has been cited as disproof of the neutral model, as if such a departure from randomness in a single gene were pertinent.
As the old expression goes, "those who are ignorant of history are doomed to repeat it." It helps a lot to be aware of the history of biology and the contributions of those who developed our current understanding. Modern science writers often fail to understand that there's not much that's new in biology these days. In this case, it's just not true that biologists were too stupid to recognize that some silent mutations weren't neutral. There was no "orthodoxy" that all silent mutations were neutral and, therefore, no orthodoxy has been overturned.

Silent mutations have no impact on the amino acid sequence of proteins and, therefore, were not expected to change their function.

Lindsay Borthwick
SEED
March 2007
The SEED article goes on to describe the results of experiments done by Kimchi-Safaty et al. (2007). They presented evidence that a cluster of three silent mutations in the MDR1 gene led to a slow down in translation and subsequent misfolding of the protein. Lindasy Bortwick then writes, "Through a series of elegant experiments, the team put to rest the idea that silent mutations were neutral." Of course, they did no such thing. They merely added one more data point to something that we already knew; namely, not all silent mutations are neutral.

Borthwick closes with,
Most fundamentally, the involvement of silent mutations in disease undermines the neutral theory of molecular evolution. This theory, posited by Motoo Kimura in the late 1960s and a powerful influence ever since, asserted that the vast majority of mutations were neutral, having no effect on the fitness of an organism, and spread through a population by chance. The fact that silent mutations are not harmless anomalies of nature means that they are not neutral. In contrast, some, if not all, silent sites must be subject to the forces of Darwinian natural selection.
The theme of the article is that neutral theory is in big trouble. This point is emphasized in the highlighted quotations that are prominently displayed on page 35 (see the two boxes above). That's totally wrong and it distorts the modern consensus among knowledgeable scientists. Neutral Theory is alive and well, thank-you very much. It can easily accommodate one more example of a non-neutral mutation.

I believe that science writers have an obligation to get the concepts right and I believe they shouldn't misrepresent the science they're supposed to be presenting in a "culturally articulate and accessible style" to a general audience. A layperson reading this article would go away with the impression that a decades old concept has just been overthrown by a single paper published in Science. That's irresponsible journalism.

Jukes, T.H. (1980) Neutral Changes Revisited. In The Evolution of Protein Structure and Function, pp. 203-219.
.
Lewontin, R.C. (1974) The Paradox of Variation. in Evolution Mark Ridley ed., Oxfrod University Press, Oxford UK.

Kimchi-Sarfaty, C., Oh, J.M., Kim, I.W., Sauna, Z.E., Calcagno, A.M., Ambudkar, S.V., and Gottesman, M.M. (2007) A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science 315, 525-528.

Kimura, M. (1968) Evolutionary rate at the molecular level. Nature 217, 624-626.

King,J.L. and Jukes,T.H. (1969) Non-Darwinian evolution. Science 164, 788-798.

18 comments:

  1. As we will see, it raises the question of whether you need to be a scientist in order to get the science right. I hope that's not true.
    Well, I would hope so too, but the fact is, the track record of non-scientist science writers is not very encouraging. This piece is not at all atypical. I think both the author of the piece, and the great majority of sceince writers, would try to characterize your comments as nit-picking. They just don't seem to see the need to sweat the details- what they want is a good story. (This is also what tends to drive scientists nuts when they're interviewed by reporters.)

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  2. I hope you realize that it's not nitpicking.

    Either Neutral Theory has been seriously challenged or it hasn't. Lindsay Bothwick says it has and I say it hasn't. That's a substantive difference of opinion, no?

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  3. Of course. What I'm saying is, science writers tend (infuriatingly) to dismiss such concern for scientific accuracy as nitpicking.

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  4. And of course, the fact that I'm not a science journalist doesn't mean you shouldn't rip into my articles if the mood strikes you.

    It's OK. No one on the web will be able to see me cry.

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  5. Although my background is in literature, I work as a science writer part-time. I've always had an interest in science, and read a great deal about biology on my own time. At my job, I write press releases about stem cell research being undertaken at the Cleveland Clinic and University Hospitals (Case Western Reserve University). I admit, it's difficult to get all the details and tell a good story. Often, my boss has me cut huge chunks, because I really do focus on details and not so much on story. I actually spend more time on doing research, making sure I understand concepts, than I do writing a piece. Fortunately, my boss has the original researchers look over my press release for accuracy. It's very helpful to get comments from the actual researchers. I think a lot of science writers would benefit if they could work more directly with research scientists.

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  6. Thanks, Larry for that nice expose of bad science reporting. I recently subscribed to SEED and have been enjoying it (mostly). When I read the Bothwick piece, I too was really annoyed. That was a couple of weeks ago (before I started my blog); I should have thought to address it there. Anyway, the quote "the involvement of silent mutations in disease undermines the neutral theory of molecular evolution" reflects the entire (false) premise of this article. SEED needs to do better if they expect me to continue as a subscriber.

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  7. There are some non-scientists who excel at writing about science (Carl Zimmer definitely comes to mind), but there are many more examples of piss-poor science writing than good science writing by non-scientists.

    Anyway, yeah, that SEED article bothered me as well. And how about a shout out for the nearly neutral theory? That's the appropriate model for the evolution of synonymous sites, or is that too much to ask?

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  8. rpm asks,

    Anyway, yeah, that SEED article bothered me as well. And how about a shout out for the nearly neutral theory? That's the appropriate model for the evolution of synonymous sites, or is that too much to ask?

    I've been defending nearly neutral theory on talk.origins and sci.bio.evolution for over a decade. I haven't blogged about it yet but I will.

    For those of you who don't know, nearly neutral theory says that there are many slightly deleterious alleles that behave to all intents and purposes as though they were neutral. It means that an allele doesn't have to be absolutely neutral in order to become fixed by random genetic drift.

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  9. Larry,

    This recent Nature Reviews Genetics article portrays neutral theory as a "theory in retreat." It seems to frame the issue using the false orthodoxy that you dislike in the SEED article. Do you agree?

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  10. PZ Myers says,

    And of course, the fact that I'm not a science journalist doesn't mean you shouldn't rip into my articles if the mood strikes you.

    Not to worry.

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  11. jsb asks,

    This recent Nature Reviews Genetics article portrays neutral theory as a "theory in retreat." It seems to frame the issue using the false orthodoxy that you dislike in the SEED article. Do you agree?

    Of course I don't agree. The authors are talking through their hat. They say in the abstract,

    However, new evidence indicates that even some synonymous mutations are subject to constraint, often because they affect splicing and/or mRNA stability.

    As you know from reading my article this is hardly new and it would not pose a threat even if it was. The statement simply reveals that the authors of the Nature review aren't good scientists. They should have read the Jukes paper (along with several dozen other papers that would have exposed the logical flaws in their arguments).

    The real question is how did this ever get published? What happened to peer review?

    I think this is a symptom of a much larger problem in modern science. The silliness over neutral theory is matched by equal silliness about alternative splicing, ev-devo, junk DNA, RNAi, adaptionism and a host of other things.
    At times it almost seems as though scientists have forgotten everything they've learned over the past 50 years. They've forgotten how to think logically.

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  12. I read that SEED article last night, and it bugged me, too. I just couldn't quite put my finger on why I didn't like the article. I thought it was because she was talking about codon bias without ever saying "codon bias" or "tRNA". I didn't know about the earlier work on not-actually-silent mutations, but I'm not surprised to hear that such work was conducted. It's also nice to discover that quote from King:
    We have, indeed, developed evidence for such exceptions ourselves, because a theory should be challenged by those who have postulated it.
    Am I alone in thinking this is a truly great thing for a scientist to say?

    Thanks for this post, I think somebody needed to point this out to SEED. I'm still quite impressed with the magazine, and I'm considering getting a subscription (price is a strong consideration - why is a subscription in Canada nearly twice the price?), but articles like this make me not want to.

    I agree with Steve Labonne - I'd expect many science writers to characterise this critique as "nit-picking", which it clearly is not. Science writing in general is a big, complex, messy topic, and I have yet to form a rigid opinion on any aspect of it - except that it should be better!

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  13. Sorry, I should have been more clear. I wondered if you agreed that the review was an example of promoting false orthodoxy--and you've answered that.

    Thanks for the post.

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  14. So Larry, Steve, et al. why not contribute an article to Seed?

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  15. trinifar,

    So Larry, Steve, et al. why not contribute an article to Seed?

    I can't speak for Steve but I'm too old to work for SEED. They're on a youth kick. Nobody over 50 need apply. I don't know what they're going to do about PZ now that he's turned 50.

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  16. I can speak for Steve. ;) 1) Nobody axed me! 2) I don't trust the editors of popular science publications, either. 3) What I would actually like to write for somewhere, if I had the opportunity is a piece about the realities of forensic DNA testing and just how much it isn't anything like on the teevee. But nobody really wants to hear that or read about the boring details of real life.

    As to actual scientific research, I think writing about that should be left primarily to practicing researchers in the field they're writing about, who have a talent for writing, like Larry and PZ. I haven't qualified for years now. But I love to see such people start blogs. And I think the best popular science books are written by practicing scientists, as well.

    But what worries me is, will they continue to have readers? Unfortunately, due to the general dumbing-down of our culture, the old-fashioned "general educated audience", of readers who are genuinely literate and intellectually curious and who have an attention span of longer than 30 seconds, seems to be shrinking all the time.

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  17. So is 50 the age where people stop referring to you as "young"?

    At least they can still fall back on "immature" in my case.

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  18. It's too bad that the focus is on the use of the term "neutral". The original paper was a fascinating example of how changes within the transcript can affect protein folding.

    Yes many have hypothesized that it could happen, but it has been rarely documented. (There was a second such paper late last year.) The observation that it can happen is the main advance of the paper and is important for those who actually study protein dynamics.

    This of course is coming from someone who studies more of the proximal questions in biology.

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