Evolution: The Missing Link in Medicine

“Nothing in biology makes sense except in the light of evolution.”

– Theodosius Dobzhansky

Evolution is arguably one of the most widely supported and powerful theories in all of biology, and potentially science as a whole. It has been a dominant explanation for over 100 years. Once genetics entered the picture in the first part of the 20th century, Darwin’s common descent and Mendel’s inheritance were improved upon, greatly expanded, and solidified into the new synthesis of evolution. Consistently verified through genetics, paleontology, geology, ecology, microbiology, and many other fields of science, evolution has become a pervasively potent field of study. It has created huge disciplinary offshoots – including evolutionary biology, evolutionary genetics, and evolutionary anthropology to name a few – and has become the theoretical foundation for all of biology.

Some people today argue that humans are no longer under evolutionary pressures, and, thus, are no longer evolving. Though this seems to make sense superficially, it is simply not true. The first issue is that humans only live about 80 years; a mere snapshot of our species’  existence. It is difficult to observe phenotypic differences as a result of biological evolution in only a few decades. That being said, scientists have found some very recent biological changes have occurred, including the altered expression of the FTO gene. The FTO gene codes for a protein that regulates appetite. While it does not “make” a person obese (genes tend to predispose, not determine), it has been correlated with obesity. The catch? It seems to have only been expressed after about 1940, according to a study published just 2 days ago. The study (which can be found here) found that, after 1942, the FTO gene showed a strong correlation with increase BMI. Why, though, would a gene that has not changed suddenly become active?

The Environment

What did change in the 1940’s? Technology. WWII offered an incredible economic boost to the US that massively increased technological enterprise and was the main contributing factor the world superpower status that the US achieved in the 40’s. As technology increased, labor decreased. After all, the main purpose of technology is to make human life simpler. When human life becomes simpler, people become more sedentary. New technology also allowed for cheaper, higher calorie, over-processed food. This one will take a while to work out. The difference could be epigenetic alteration, novel environmental stimuli, or even another gene interacting with FTO. While more testing will be needed to show exactly what happened in the early 40’s that altered FTO expression, the fact that something did occur, likely stemming from environmental changes, still remains. Biological evolution doesn’t have to be the changing of DNA sequence; that is far too simplistic. Anytime phenotypic or genotype ratios change on a species-wide level, evolution is occurring. No population is in Hardy-Weinberg equilibrium, and no population ever will be. Human wills continue to evolve biologically. While cultural evolution has exceedingly outpaced biological evolution, giving the mirage that biological evolution has “stopped,” the truth is that culture can either augment or stagnate biological evolution, depending upon the situation. A cultural change to drinking more milk may augment lactase persistence (and in fact, it did), while a cultural propensity to live in climate controlled housing year-round may slow other aspects of biological evolution. Nature doesn’t necessarily control natural selection; more broadly, the environment (cultural or natural) mediates evolution.

So, why is evolution important in medicine? Sure, doctors need to understand things like microbial evolution and how it plays a role in infectious diseases, but what about human evolution? How can a knowledge of human evolution impact medicine?

Cultural evolution has rapidly and drastically altered the human environment, thus changing how the human species evolves. More importantly, our environments have changed so aggressively that our bodies cannot keep up. (Before I go on, I have to make something clear. I am not a proponent of the Paleo Diet; if you’d like to know why, check out this post.) This means our bodies are often best adapted to the environments of the past (though these vary drastically). This has given rise to what are sometimes referred to as “mismatch diseases.” The list is extensive, but includes maladies such as atherosclerosis, heart disease, type-2 diabetes, osteoporosis, cavities, asthma, certain cancers, flat feet, depression, fatty liver syndrome, plantar fasciitis, and irritable bowel syndrome, to name a few. Some of these may not be actual mismatch diseases, but many of them likely are. Furthermore, many of these illnesses feed off one another, creating a terrible feedback loop. 100 years ago you’d likely die from an infectious disease; today, most people in developed nations will die from heart disease, type-2 diabetes, or cancer.

These diseases don’t have to be essential baggage of modernity. Anthropologists and (and some intrepid human evolutionary biologists) study modern day hunter-gatherer societies in order to glean information about the nature of our species pre-Neolithic Revolution. It’s important to note that these are not perfect models (cultural and biological evolution has still occurred in these hunter-gatherer societies), but are the best available. Interestingly, modern day hunter-gatherers don’t suffer from many of these mismatch diseases (This effect can’t be explained by longevity; hunter-gatherers regularly live into their late 60’s and 70’s. Though unusual to many of us, their lives aren’t as brutish as they are often portrayed). Diseases such as type-2 diabetes, hypertension, heart disease, osteoporosis, breast cancer and liver disease are rare among the societies. What’s more, myopia (near-sightedness), asthma, cavities, lower back pain, crowded teeth, collapsed arches, plantar fasciitis, and many other modern ailments are exceedingly rare. So what’s different? The easy answer is their diet, lifestyle, and environment. The difficult answer involves elucidating the physiological importance of certain social norms and biochemical processes of differing diets. Some very exciting work is beginning to arise in this field, dubbed “evolutionary medicine.”

Modern medicine and medical research focuses largely on treating problems, i.e., drugs and procedures that alleviate symptoms after the disease has manifested. While the cause is noble, and indeed necessary, it’s not enough. The childish logic of medical research creates a cycle of sickness-treatment that, in 2012, totaled almost $3 trillion in healthcare costs. Furthermore, the sedentary and Epicurean lifestyle in which many Americans live willingly feeds this cycle; among the less privileged, necessity feeds this cycle through the inability to afford healthy food, limited access to health education, and a sociocultural feedback loop that breeds its own vicious cycle.

There will likely never be a drug that can cure cancer (of which there are thousands of variants that can even differ between individuals who have the same variant), heart disease, type-2 diabetes, or many of the other previously mentioned noninfectious diseases. The rationale is akin putting water in your car’s gas tank and hoping additives will make it work as efficiently as gasoline. The car was built to run off gasoline. Similarly, your body has evolved to not eat an excessive amount of salts, carbs, and sugars (of which the different types, particularly glucose and fructose, do not have the same biochemical effects during digestion), sit for extended periods of time, wear shoes (particularly those with arch support; a common misconception is that arch support is good for you when, in fact, it weakens the muscles of the arch, leading to ailments such as collapsed arches and plantar fasciitis), read for several hours at a time, chew overly processed food, or many of the other things that people in developed nations commonly do, often times see as a luxury.

Modern medicine needs a paradigm shift. Funding needs to support not only treatments, but also investigations into prevention. The medical cause of diabetes may be insulin resistance, but what causes insulin resistance and how can we prevent it? Sugar may cause cavities, but what can do to prevent this? Shoes, even comfy ones, may cause collapsed arches, but how do we prevent this? The immediate response may be that this sort of prevention cannot be attained without abandoning modern technology all together. However, this isn’t the case, and it’s not the argument I’m trying to make. Research should focus on a broad range of interacting variable, including diet, work environment, school environment, and other aspects evolutionarily novel environments. Only after research from this evolutionary perspective takes place can constructive conversations and beneficial environmental changes occur. We don’t have to abandon modern society to be healthy; we just need to better understand how our lifestyle affects our bodies. Items such as smoking and alcohol are already age limited and touted as dangerous to health. Is junk food, particularly soda, any different? We don’t put age regulations on cigarettes or alcohol to protect bystanders. Instead, these regulations protect children who cannot be relied upon to make proper choices in their naivety. Should soda be under these same constraints?

If medicine and medical research does not undergo this paradigmatic shift and incorporate an evolutionary perspective, the outcome does not bode well for us. Medical costs will continue to rise with little room for improvement and greater opportunities for socioeconomic factors to play into the quality of healthcare available. This ad hoc treatment approach to medicine is not sustainable, and is not the best we can do.

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Multiplex Automated Genome Engineering: Changing the world with MAGE

Humans have evolved a most unique mastery of toolmaking through advanced technology. As an extension of our biological bodies, technology has loosened the grip of natural selection. This is particularly true in the field of biomedicine and genetic engineering. We have the ability to directly alter the blueprint of life for any purpose we wish. Beginning in the 1970’s with the creation of recombinant DNA and transgenic organisms, genetic engineering has offered scientists the ability to study genes on a level that may not have seemed possible at the time. The field has provided a wealth of knowledge as well as practical implications, such as knockout mice and the ability to produce near-endless amount of human insulin for diabetics.

As of 2009, multiplex automated genome engineering (MAGE) has ushered in a new branch of genetic engineering – genomic engineering. We are no longer restricted to altering single genes, but rather are able to alter entire genomes by manipulating several genes in parallel. This new ability, brought about by MAGE technology, allows for nearly endless applications that stretch well beyond medicine or industry; agriculture, evolutionary biology, and conservation biology will benefit tremendously as MAGE technology progresses. Genetic engineering advancements such as MAGE are poised to revolutionize entire fields of science, including synthetic biology, molecular biology, and genetics by offering faster, cheaper, and more powerful methods of genome engineering.

Homologous Recombination

Genetic engineering underwent a revolutionary change in the 1980’s, largely due to the pioneering work of Martin Evans, Mario Capecchi, and Oliver Smithies. Evans and Kauffman were the first to describe a method for extracting, isolating, and culturing mouse embryonic stem cells. This laid the foundation for gene targeting, a method that was independently discovered by both Oliver Smithies and Mario Capecchi. Mario Capecchi and his colleagues were the first to suggest mammalian cells had the machinery capable for homologous recombination with exogenous DNA. Smithies took this a step further, demonstrating targeted gene insertion using the β-globin gene. Ultimately, the combined work of Evans, Smithies, and Capecchi on homologous recombination earned them the Nobel Prize in Physiology or Medicine in 2007. The science of homologous recombination has allowed for many scientific discoveries, primarily through the creation of knockout mice.

Homologous recombination works under many of the same principles are chromosomal recombination in meiosis, wherein homologous genetic sequences are randomly exchanged. The difference lies in the fact that homologous recombination works with exogenous DNA and on a gene level rather than chromosomal level.

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The method works by using a double stranded genetic construct with flanking regions that are homologous to the flanking regions of the gene of interest. This allows for the sequence in the middle, containing a positive selection marker and new gene, to be incorporated. The positive control should be something that can be selected for, such as resistance to a toxin or a color change. Outside of one of the flanking regions of the construct should lie a negative selection marker; the thymidine kinase gene is commonly used. If homologous recombination is too lenient, and the thymidine kinase gene is incorporated into the endogenous DNA, it can be detected and disposed of. This is to prevent too much genetic information from being exchanged.

Using this method, knockout mice can be created. A knockout mouse is a mouse that is lacking a functional gene, allowing for elucidation of the gene’s function. Embryonic stem cells are extracted from a mouse blastocyst and introduced to the gene construct via electroporation. The successfully genetically modified stem cells are selected using the positive and negative markers. These are isolated and cultured before being inserted back into mouse blastocysts. The mouse blastocysts can then be inserted into female mice, producing chimeric offspring. These offspring may be mated to wild-type mice. If the germ cells of the chimeric mouse were generated from the modified stem cells, then the offspring will be heterozygous for the modified gene and wild-type gene. These heterozygous mice can then be interbred, with a portion of the offspring being homozygous for the modified gene. This is the beginning of a mouse line with the chosen gene “knocked-out.”

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Multiplex Automated Genome Engineering Process

The major drawback of the previously described method of “gene targeting” is the inability to multiplex. The process is not very efficient, and targeting more than one gene becomes problematic, limiting homologous recombination to single genes. In 2009, George Church and colleagues solved this issue with the creation of multiplex automated genome engineering (MAGE). MAGE technology uses hybridizing oligonucleotides to alter multiple genes in parallel. The machine may be thought of as an “evolution machine,” wherein favorable sequences are chosen at a higher frequency than less favorable sequences. The hybridization free energy is a predictor of allelic replacement efficiency. As cycles complete, sequences become more similar to the oligonucleotide sequence, increasing the chance that those sequences will be further altered by hybridization. Eventually, the majority of endogenous sequences will be completely replaced with the sequence of the oligonucleotide. This process only takes about 6-8 cycles.

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After the E. coli cells are grown to the mid log phase, expression of the beta protein is induced. Cells are chilled and the media is drained. A solution containing the oligonucleotides is added, followed by electroporation. This step is particularly lethal, killing many of the cells. However, the cells are chosen based on positive markers (optional, but increases efficiency) and allowed to reach the mid-log phase again before repeating the process. Church and his colleagues have optimized the E. coli strain EcNR2 to work with MAGE. EcNR2 contains a plasmid with the λ phage genes exo, beta, and gam as well as being mismatch gene deficient. When expressed, the phage genes will help keep the oligonucleotide annealed to the lagging strand of the DNA during replication, while the mismatch gene deficiency prevents the cellular repair mechanisms from changing the oligonucleotide sequence once it is annealed. Using an improved technique called co-selection MAGE (CoS-MAGE), Church and colleagues created EcHW47, the successor to EcNR2. In CoS-MAGE, cells that exhibit naturally superior oligo-uptake are selected for before attempting to target the genes of interest.

MAGE technology is currently in the process of being refined, but shows incredible promise in practical applications. Some of the immediate applications include the ability to more easily and directly study molecular evolution and the creation of more efficient bacterial production of industrial chemicals and biologically relevant hormones. Once the technique has been optimized in plants and mammals, immediate applications could be realized in GMO production and creation of multi-knockout mice that will give scientists the ability to study gene-gene interactions on a level previously unattainable. A more optimistic and perhaps grandiose vision could see MAGE working towards ending genetic disorders (CRISPR technology, an equally incredible genomic editing technique, may beat MAGE there) and serving as a cornerstone technique in de-extinction. The ability to alter a genome in any fashion brings with it immense power. The possibilities for MAGE are boundless, unimaginable, and are sure to change genomic science.

For more information on Homologous recombination, see:

http://www.bio.davidson.edu/genomics/method/homolrecomb.html

For more information on MAGE, see:

Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M. (2009). Programming cells by multiplex genome engineering and accelerated evolution. Nature, 460(7257), 894-898.

Wang, H. H., Kim, H., Cong, L., Jeong, J., Bang, D., & Church, G. M. (2012). Genome-scale promoter engineering by coselection MAGE. Nature methods, 9(6), 591-593.

For more information on CRISPR (which I highly recommend; it’s fascinating), see:

https://www.addgene.org/CRISPR/guide/