In contrast to polymorphisms, rare DNA variants—those much less frequent in humans than SNPs—can have large effects on the risk of developing some common diseases. For example, a number of different loss-of-function mutations, each individually rare, have been found to increase greatly the predisposition to autism and schizophrenia. Many of these are de novo mutations, which arose spontaneously in the germ-line cells of one or the other parent. The fact that these mutations arise spontaneously with some frequency could help explain why these common disorders—each observed in about 1% of the population—remain with us, even though the affected individuals leave few or no descendants. These rare mutations may arise in any one of hundreds of different genes, which could explain much of the clinical variability of autism and schizophrenia. Because they are kept rare by natural selection, most such variants with a large effect on risk would be missed by genome-wide association studies.
Now that DNA sequencing has become fast and inexpensive, the most efficient and cost-effective way to identify these rare, large-effect mutations is by sequencing the genomes of affected individuals, along with those of their parents and siblings as controls.
Reverse Genetics Begins with a Known Gene and Determines Which Cell Processes Require Its Function
As we have seen, classical genetics starts with a mutant phenotype (or, in the case of humans, a range of characteristics) and identifies the mutations, and consequently the genes, responsible for it. Recombinant DNA technology has made possible a different type of genetic approach, one that is used widely in a variety of genetically tractable species. Instead of beginning with a mutant organism and using it to identify a gene and its protein, an investigator can start with a particular gene and proceed to make mutations in it, creating mutant cells or organisms so as to analyze the gene’s function. Because this approach reverses the traditional direction of genetic discovery—proceeding from genes to mutations, rather than vice versa—it is commonly referred to as reverse genetics. And because the genome of the organism is deliberately altered in a particular way, this approach is also called genome engineering or genome editing. We shall see in this chapter that this approach can be scaled upward so that whole collections of organisms can be created, each of which has a different gene altered.
There are several ways a gene of interest can be altered. In the simplest, the gene can simply be deleted from the genome, although in a diploid organism, this requires that both copies—one on each chromosome homolog—be deleted. Although somewhat counterintuitive, one of the best ways to discover the function of a gene is by observing the effects of not having it. Such “gene knockouts” are especially useful if the gene is not essential. Through reverse genetics, the gene in question (even if it is essential) can also be replaced by one that is expressed in the wrong tissue or at the wrong time in development; this type of manipulation often provides important clues to the gene’s normal function. For example, a gene of interest can be modified to be expressed at will by the experimenter (Figure 8–52). Finally, genes can also be engineered so that they are expressed normally in most cell types and tissues but deleted in certain cell types or tissues selected by the experimenter (see Figure 5–66). This approach is especially useful when a gene has different roles in different tissues.
It is also possible to make subtler changes to a gene. It is sometimes useful to make slight changes in a protein’s structure so that one can begin to dissect which portions of a protein are important for its function. The activity of an enzyme, for example, can be studied by changing a single amino acid in its active site. It is also possible, through genome engineering, to create new types of proteins in an