by Frank Ryan
In the twinkling of an eye our puffing engine has reappeared before us, ready to roll, and we hop on board, tootle the whistle, and head eastward at a rattling rate.
“Keep your eyes peeled for a red light up ahead.”
After what could have been quite a few miles, you spot it. A pinpoint, red glow in the distance. “It's in the track, to our right.”
“Yes. It would have to be in the new daughter copy.” I explain that the track closest to us is called the “sense” track, because it is the original. The daughter copy on the other rail is the “antisense” track. The genetic machinery reads this while traveling in the opposite direction. I slow the engine to a halt so we can see what the red light indicates. “Look at the sleepers.”
You get down onto your haunches to have a closer look. At first you can't make out anything wrong. The split in the sleeper appears to be as before, with the short section to the left, then the short section to the right. Then you gasp. “The one on the left is a C, so the other half of the sleeper should have been a G. But it isn't. It's an A.”
“So?”
“The copying mechanism has made a mistake.”
“Yes.”
“So this is…a mutation?”
“Yes, it is. To be precise, it's what is called a point mutation, which means a single nucleotide has been miscopied. But if and when the anti-sense strand copies itself, the mutated nucleotide will attract a thymine to attach to it—in other words the mutation will now be fixed into the double helix. The mutation will, in this way, perpetuate itself. If this were to happen during the formation of the germ cells, the sperm or the ovum, that germ cell would carry the mutation into the genome of the new generation.”
“Are these mutations common?”
“Much more common than one might think. But thankfully there are compensating mechanisms in that moving cloud that will usually recognize and correct them. But mutations do get through from time to time.”
“And this will cause disease?”
“Most mutations don't cause disease. They only do so if they affect a part of the DNA that fulfills an important role in the offspring's internal genetics or they seriously affect the coding of a protein-coding gene.”
In the early years of the twentieth century, a Dutch botanist, Hugo de Vries, made the conceptual breakthrough that Mendel's discrete packages of hereditary information could be changed by mutations. Amazingly he did so before we knew anything about the actual structure of DNA or what constituted a gene. As we have just witnessed, a mutation is an error in the nucleotide sequence made during the copying of DNA. Mutations can arise, albeit rarely, during the normal process of DNA copying. They can be induced at a much higher rate if the DNA replication is damaged by external influences, such as exposure to toxic chemicals or excessive doses of radiation.
There are many different types of mutation. What we have witnessed is one of the simplest, in which a single nucleotide has been substituted—a point mutation. A so-called “frame-shift mutation” would result from the simple deletion of a nucleotide. If we imagine what this would do to the succeeding triple codons we realize that it would interrupt all the triplet sequences following it, and thus would play havoc with the translated protein. Even a point mutation in a protein-coding gene might result in a changed amino acid in the coded-for protein. This is what causes sickle-cell anemia. In this case, the mutation replaces what should be an adenine in the beta-globin gene for a thymine. When this translates to the beta-globin protein, the amino acid glutamic acid is replaced by valine. This makes the abnormal hemoglobin in the red cells that causes the disease. If the offspring gets just one copy of the mutated gene, they suffer a milder form of the disease that incidentally protects them from malaria. If they get a double dose of the mutated gene—in other words, if they get a mutated gene from both their parents—they get a severe form of the disease that can be fatal early in life. Mutations affecting the cells of tissues and organs in the body, as opposed to the germ cell, are an integral part of the underlying causes of many different forms of cancer.
There are a few additional terms I need to explain to present an outline of basic genetics. Other than the sex chromosomes, X and Y, we inherit 22 non-sex-connected chromosomes from each of our parents. These are called “autosomes.” This means that we all, both males and females, inherit two copies of every gene that is found on the autosomal chromosomes, which amounts to the bulk of our genes. When a mutation affects an autosomal gene during the formation of the ovum or sperm it will only affect one of the two copies in the offspring. If the remaining normal copy of the gene is enough to supply the body's biochemical needs, there will be no upset in the internal chemistry—no clinical disease. This type is called a “recessive” gene mutation. But sometimes just one bad gene is enough to give rise to serious disturbance in the internal chemistry, despite the fact the other gene is normal. This is called a “dominant” gene mutation. When a mutation, whether dominant or recessive, gives rise to a disease, this is referred to by doctors as “an inherited disorder of metabolism” or “an inborn error of metabolism.”
Many medical conditions arise from dominant genes; for example, Huntington's disease, a condition in which the affected person may develop a progressive cerebral deterioration later in life. The inheritance of one recessive gene isn't enough to cause an inherited disease of metabolism, but if both parents are carrying one copy of the same recessive mutated gene, then there is a one-in-four chance of the offspring being unlucky enough to inherit mutated versions of the gene from both parents. Since there is no normal copy, this will then give rise to disease.
One in every 2,500 babies born to Caucasian parents suffers from cystic fibrosis, making it one of the most common hereditary diseases. It is caused by a variety of mutations affecting a regulator gene, which is known as the cystic fibrosis transmembrane regulator gene, or CFTR, located in the region q31–32 of human chromosome 7, and which codes for an ion channel involved in transport across membranes. Cystic fibrosis is perhaps the most familiar example of an autosomal recessive condition. There are many other recessive genetic disorders that might potentially be cured by the addition of a single “normal” gene, and these conditions, including cystic fibrosis, are the subject of intensive current investigation aimed at “gene therapy.”
Another pattern of mutation gives rise to a sex-associated recessive condition. Females have two of the sex-associated chromosomes called “X” chromosomes, while males only have one X, always inherited from the mother. This means that a recessive gene that happens to be carried on the X chromosome will usually have no serious effects in females but it will behave like a dominant gene if inherited by a male. A sex-linked recessive mutant gene is the cause of hemophilia, a condition that ravaged some of the royal houses of Europe. It is also the cause of the red-green color blindness that affects between 7 percent and 10 percent of men, as well as several types of muscular dystrophy.
Such single-gene mutations will usually be inherited along Mendelian lines, such as the dominantly inherited achondroplasia and Huntington's disease, the recessively inherited cystic fibrosis, and the sex-chromosome-linked disorders. To date, geneticists have identified more than 5,000 single-gene disorders in humans caused by mutations. Some mutations can change the number of chromosomes, as in Down's syndrome, or delete, duplicate, fragment, or otherwise damage the structure of chromosomes, giving rise to a variety of syndromes. As mentioned above, mutation is also a common feature of cancers, which usually arise in fully developed tissues long after embryogenesis. Other chromosomal abnormalities affect the germ cells, where they give rise to a wide range of disorders including aberrant embryological development, with resultant congenital abnormality, as well as a great many inborn errors of metabolism. In all such cases, a clear understanding of the genetic cause, or causes, is the basis for medical prevention and therapy.
The medical approach to mutation includes genetic counseling. For example, enabling couples at risk
of particular disorders to have essential information so they can make their own decisions on matters of reproduction, and public education about the risks of increasing maternal age; avoidance of risk factors such as irradiation of the germ cells and fetus, caution with respect to drug and chemical exposure, such as thalidomide; and vaccination against rubella. Newer measures, such as preimplantation genetic diagnosis, involve the genetic screening of the fetus at the 16- or 32-cell stages, followed by the selection and implantation of healthy embryos. This requires a genetic abnormality that is predictable, together with the availability of a suitable screening test in isolated embryological cells. It not only reduces the risk of severe abnormality in children in very high-risk circumstances but also removes the mutation, and thus the risk pedigree, in future generations. There are, of course, important ethical and moral principles involved in such therapy for both doctor and patients in what essentially amounts to a positive form of eugenics.
Cancer is another arena in which intensive study of the mutated genes offers the hope of developing more efficient therapies. Here the genetic abnormalities are more complex than in the inherited diseases and very often involve multiple mutations as well as important links to environmental factors. At the genetic level, cancer involves a series of steps that involve multiple mutations that deregulate regulatory pathways. New lines of research suggest that these mutations must cooperate with each other for the cancer to develop, so research aimed at determining the precise nature of the cooperating mutations and the regulatory pathways they affect is a major challenge. The decoding of the human genome has highlighted the genetic alterations that underlie cancers in such unprecedented detail that it has led two American oncologists, Vogelstein and Kinzler, to declare that “cancer is, in essence, a genetic disease.”
Some 15 to 20 percent of women with breast cancer have a family history of the condition, and 5 percent of all breast cancers have been linked to mutations in the genes BRCA1 and BRCA2. Geneticists can further predict that women who carry these mutations have an 80 percent risk of developing breast cancer during their lifetime. There are various options that help to reduce the risk, including prophylactic ovariectomy, regular breast screening, and the potential of preemptive surgery.
In 2006, a systematic multi-center American study pioneered the screening of more than 13,000 genes taken from human breast and colon cancer cells. Given the “normal” human genome, they were in a position to compare the genes they found in the two cancers with the normal, revealing that individual tumors accumulate an average of 90 mutant genes. It seems that a much smaller number of these actually play a part in the cancer process; in their estimation, perhaps 11 mutations for each of breast and colon cancer. Encouraged by these findings, the US National Institutes of Health is drawing up an atlas of cancer genomes—The Cancer Genome Atlas Project, or TCGA. The aim is to decode the genomes of every human cancer and, by comparing these to the normal, extrapolate the genetic abnormalities that underlie all cancers. A pilot study has begun with cancers of the lung, brain, and ovaries. This is far from pie in the sky research; already cancer is being forced back on many different fronts, and today some forms of cancers are eminently treatable by surgery, focused radiotherapy, and chemotherapy or immunotherapy, so that what might formerly have been a death sentence has become more a chronic but controllable ailment.
Of the three main activities involved in scientific research—thinking, talking, and doing—I much prefer the last and am probably best at it. I am all right at the thinking, but not much good at the talking.
FREDERICK SANGER
In the late 1960s, I was privileged to be a medical student at the University of Sheffield. Watson and Crick were still relatively young men, their discovery having been made just fifteen or sixteen years earlier. I can remember my own sense of wonder as our teachers explained the structure of DNA and the elegance with which its four-letter code translated to proteins. We had lectures on genetics in which we learned how mutation was a major step in our understanding of many different hereditary diseases, including the so-called “inherited errors of metabolism.” We also had lectures on the importance of the same discoveries to the sister discipline of evolutionary biology. I can recall the prevailing sense of excitement that came with the feeling that the biological and medical sciences were entering a new paradigm, based on the growing understanding of DNA and its molecular extrapolations, an understanding that clearly had implications not merely for biological scientists and doctors, but for all of humanity. But at that stage many important questions remained to be answered.
One very obvious question was how did the fertilized egg, or “zygote,” develop into the complex wonder of a human baby? How could this extraordinary chemical, DNA, store not only the heredity of the individual but also the instructional blueprint that was necessary for the single cell of the zygote to give rise to the developing embryo, with its wide range of different cells and tissues and organs that went into making the future baby?
While much was known about the tissue changes within the embryo, little was actually known of the relevant genetics at this time. The work of the scientists at the Pasteur Institute in France offered us the first glimpse into this quandary: they had pioneered our understanding of how a gene is activated by switching on its “promoter” sequence and how it is inactivated by switching off the promoter. This was the first step toward what we now call genetic “regulation.”
Back then we also knew that the cells that make up the different tissues and organs in the human body—such as the brain cells or the white cells that fight off infection in the circulating blood or the cells that make up the kidney, or liver, heart or lung—all contained exactly the same DNA in their nuclei. The differences in structure and function between these cells, and thus the makeup of the various tissues and organs, must somehow involve differences in the expression of genes. This provoked two new questions: Was the difference brought about by specific genes that were only switched on in specific organs, or was it brought about by different profiles and timing of the expression of the same genes?
The questions did not stop there.
Whatever the explanation, whether special genes for particular cells, or different profiles of expression of the same genes, there had to be a system that decided what gene, or what profile, would be expressed in the different cells, tissues, and organs. This must be a key element in the planning and regulation of the developing human embryo—and very likely there would be very similar patterns of regulation of embryogenesis in all animals—and maybe plants as well.
We might recall here Sydney Brenner, who came to work with Crick at the Cavendish Laboratory on the translation of genes to proteins. In 1973, when still employed by the MRC Laboratory in Cambridge, Brenner published a paper that addressed this very subject. It opened with the lines, “How genes might specify the complex structures found in higher organisms is a major unsolved problem in biology.” He explained that by now many of the molecular mechanisms previously shown in microbes were found to exist in much the same form and function in eukaryotic cells—the nucleated cells of animals and plants. The genetic code was universal, and the translation of that code to the mechanisms of protein synthesis appeared to be equally universal. Meanwhile, “Although there are many theories suggesting how the [DNA of higher organisms might control such] complex genetic regulation, the problem is still opaque.” Brenner chose a new model system for research into how animal genes were controlled and organized. In his paper he introduced his new model, a minuscule round-worm, Caenorhabditis elegans, which was just a millimeter long and a common inhabitant of temperate soil environments. C. elegans had a number of attractive properties for this type of research. It was non-parasitic, so it wouldn't infect laboratory workers; it was very simple in structure, with the entire worm comprising just 959 cells; it could easily be bred; it was conveniently transparent so one could peer inside it through a microscope; it had a tiny genome comprising just five pairs of au
tosomes and one pair of sex chromosomes; and it comprised two sexes—hermaphrodite and male. In a nutshell, it presented geneticists with an ideal model experimental animal, being easy to breed, safe to store in large numbers, with a sexuality and a genetics that could easily be manipulated.
In his paper, Brenner showed how he had used experimentally induced mutations in some 300 of the worm's genes to show how these genes contributed to the worm's biological makeup and behavior. But even in a creature as simple as the worm, the genetics proved to be more complex than Brenner had imagined. A staggering 77 different genes were involved in its simple wriggly movements. Nevertheless, study of the worm soon confirmed his choice of experimental model. Here was a new experimental model capable of figuring out what genes did and in particular how they regulated the mysterious and profound changes that took place during embryological development, when those extraordinary pluripotent cells of the early embryo began the processes of change that would ultimately give rise to the cells of the many different body tissues and organs.
Brenner's model proved to be an inspired choice. It was taken up in many different scientific centers, and as knowledge grew, the C. elegans, which itself complemented the earlier fruit fly research, was complemented by pioneering explorations of gene function and gene regulation in fish, frogs, lancelets, and mammals in the form of mice—as well as a growing variety of plants.
The human body contains about 200 different types of cell, formed into limbs, tissues, and organs, each specialized to perform distinct functions. For the zygote to develop into all of these, it must begin its life as a “totipotent” cell—a cell that can differentiate into every possible human tissue, including the placenta as well as the developing fetus. The first differentiation is from this stage of totipotent to “pluripotent” cells—which means cells with multiple but not total potency. The pluripotent cells are the cells that now give rise to the more complex shapes and cellular differentiation that will begin to fashion the distinct tissues and organs. These same pluripotent cells, also referred to as “stem cells,” remain with us for the rest of our lives, replacing damaged tissues in the constant recycling that is necessary for normal physiological functioning and health. For such a remarkable transformation to occur in the embryo with such predictable precision, each cell must know its own fate. This is determined by a carefully controlled bureaucracy of genetic elements including epigenetic regulation, which we shall come to in a subsequent chapter, as well as entities known as “regulatory genes.”