The Human Knock-out: looking for non-working genes

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The word "knock-out" in biology is used for lab animals like mice, for example, when one of their genes is silenced in order to study the effects of not having that gene. Silencing a gene (or knocking it out, hence the nomenclature "knock-out mouse") means that gene is no longer producing the protein it codes for. This is a condition sought for in situations where you have to test for a drug and hence the first step is to reproduce the genetic condition that caused the disease.

Mice are often "humanized", i.e. genetically engineered to carry human genes so that the experiment can be a better model for drug or therapy testing. Unfortunately, even when humanized, mice or lab animals in general are poor models for humans. When things don't work out in an animal model, we know that the experiment should not be carried out on to humans, but when on the other hand things go well in an animal experiment, there is no guarantee that it will work on humans too.

"Human chip" technology is a very promising solution, as it would bypass the need of animal testing for drug discovery. The idea is to have cell cultures from different organs on a "chip" the size of a smart phone. Lung, liver, kidney chips have already been designed and tested, but lately there has been an even further advance in making the chips part of a network connected by "blood vessels": Athena (Advanced Tissue-engineered Human Ectypal Network Analyzer) is an ongoing project to see how four organ chips (liver, lung, heart and kidney), connected by tubed filled with artificial blood, can effectively simulate a human body for drug testing and toxin screening. Athena, also dubbed the "desktop human" as given its size it would conveniently sit on a desktop, is a $19 million dollars project that will be built in the next five years.
You can read the full story here.

Athena, however, only has four organs and is still poor surrogate of the human body. The ideal solution would be to have human knock-outs to study the true effect of drugs, which of course is a little unethical to pursue. Unless human knock-outs already exist in nature. Well, guess what? They do, and they are far more common than we originally thought: on average every person has about 20 inactivated genes [1]. Wait, it gets better. Because, you may wonder, if they are so common, how come we never noticed? The ~20 inactivated genes must have some effects and/or symptoms, right?

Not necessarily. Yes, that's the most amazing thing: how robust our DNA is. People can have inactivated genes and still be healthy. It doesn't always happen, yet there are some cases when deficient gene copies are somehow compensated by other genes. And that's exactly why studying these human knock-outs is so relevant: we need to understand how people can stay healthy even when lacking important genes, as this can give new insight in drug discovery and therapy development.

In [1], MacArthur et al. screened close to 3,000 variants predicted to cause loss of gene function from 185 human genomes. Then challenge is to distinguish the "true" loss of function variants from sequencing errors. The researchers designed a "filter" to distinguish the "true" variants from the artificial errors. To me, the most striking discovery they made is that loss of function doesn't work as an "on/off" switch, rather, it can lead to a range of possible scenarios:

"Homozygous inactivation of a gene can have a range of phenotypic effects: At one end of the spectrum are severe recessive disease genes, while at the other end are genes that can be inactivated with- out overt clinical impact, referred to here as LoF- tolerant genes. Clinical sequencing projects seeking to identify disease-causing mutations would benefit from improved methods to distinguish where along this spectrum each affected gene lies [1]."

Jocelyin Kaiser wrote a nice article on Science [2] on the recent developments of this type of research: the plan is to sequence the genome of many more "healthy" people, find what genes they have inactivated, and then study their clinical characteristics. Some of these loss of function variations may end up being beneficial, as is the case for PCSK9, for example: the gene encodes for the homonymous enzyme, which has been associated with high cholesterol. As it turns out, individuals who carry loss of function mutations in this gene have low cholesterol and a significantly reduced risk of stroke and heart disease [3].

[1] MacArthur, D., Balasubramanian, S., Frankish, A., Huang, N., Morris, J., Walter, K., Jostins, L., Habegger, L., Pickrell, J., Montgomery, S., Albers, C., Zhang, Z., Conrad, D., Lunter, G., Zheng, H., Ayub, Q., DePristo, M., Banks, E., Hu, M., Handsaker, R., Rosenfeld, J., Fromer, M., Jin, M., Mu, X., Khurana, E., Ye, K., Kay, M., Saunders, G., Suner, M., Hunt, T., Barnes, I., Amid, C., Carvalho-Silva, D., Bignell, A., Snow, C., Yngvadottir, B., Bumpstead, S., Cooper, D., Xue, Y., Romero, I., , ., Wang, J., Li, Y., Gibbs, R., McCarroll, S., Dermitzakis, E., Pritchard, J., Barrett, J., Harrow, J., Hurles, M., Gerstein, M., & Tyler-Smith, C. (2012). A Systematic Survey of Loss-of-Function Variants in Human Protein-Coding Genes Science, 335 (6070), 823-828 DOI: 10.1126/science.1215040

[2] Kaiser, J. (2014). The Hunt for Missing Genes Science, 344 (6185), 687-689 DOI: 10.1126/science.344.6185.687

[3] Cohen, J., Pertsemlidis, A., Kotowski, I., Graham, R., Garcia, C., & Hobbs, H. (2005). Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9 Nature Genetics, 37 (2), 161-165 DOI: 10.1038/ng1509