Mutation, gene expression and cancer

The kinds of mutation, and what each does to a protein

A mutation is a change in the base sequence of DNA. The two commonest kinds arise as copying errors during replication. They are set out below with their consequences for the polypeptide. Prerequisite: the genetic code and transcription.

A substitution replaces one base with another and affects one triplet only. Three outcomes are possible. If the new triplet codes for the same amino acid, which happens often because the code is degenerate and triplets sharing an amino acid usually differ in the third base, the mutation is silent and the polypeptide is unchanged. If it codes for a different amino acid the mutation is missense, and the effect ranges from none to complete loss of function, depending on where in the protein the changed residue sits. If it produces a stop codon the mutation is nonsense, translation ends early, and the truncated polypeptide is usually non-functional.

An insertion or a deletion within a coding sequence, of a number of bases not divisible by three, shifts the reading frame, because the code is non-overlapping and read in threes from a fixed start. Every triplet downstream is read from a new position, which is a frame shift, so the amino acid sequence after the mutation differs from the original and a stop codon usually arises early by chance. The effect depends on the position of the change within the transcript and on how the transcript is processed: a frame shift near the start of a coding sequence usually gives a non-functional protein, while one near the end may not. A deletion of three bases removes one amino acid and shifts nothing, which is why the commonest cystic fibrosis allele, a deletion of three bases removing a single phenylalanine, produces a protein that is complete but misfolds: the cell's quality control holds it back in the endoplasmic reticulum and degrades most of it, so almost none reaches the membrane.

Describe the drawing

A comparison of four versions of the same short messenger RNA sequence, written as four codons in a row with the amino acid each one specifies printed underneath. The first row is the original, AUG CCU GAA UUC, giving methionine, proline, glutamic acid and phenylalanine. In the second row the third codon has become GAG, which still gives glutamic acid. In the third it has become GUA, giving valine instead. In the fourth it has become UAA, a stop codon. Below a dashed line, a fifth row shows a single base deleted, after which the codons no longer line up with those above and every one that follows is read differently.

A human inherits roughly sixty to seventy new point mutations that neither parent had, most of them from the father, since sperm come from many more rounds of division than eggs do; almost all land in non-coding DNA and do nothing. The rate rises with exposure to mutagens: ultraviolet light, which links adjacent thymine bases; ionising radiation, which breaks strands; and chemicals such as the benzopyrene in tobacco smoke, which distorts the helix so that polymerase misreads it.

Chromosome mutations: changes to structure rather than sequence

Everything in the section above is a gene mutation: a change to the base sequence of one gene, affecting one triplet or a few. A chromosome mutation is a change on a far larger scale, to the structure of a chromosome or to the number of chromosomes in a cell, and it can lose, repeat or move hundreds of genes at once. The bases inside a moved section may be exactly as they were. What has changed is where the section sits, how many copies of it the cell has, or which way round it lies. Changes in number arise by non-disjunction in meiosis and are taught with meiosis; this section deals with structure, and Pearson Edexcel Biology B examines it through translocations.

A structural change begins with breaks in the DNA of a chromosome that the cell's repair enzymes rejoin wrongly. Four outcomes are recognised.

In a reciprocal translocation two non-homologous chromosomes each break, and the broken-off pieces swap places. Nothing is lost and nothing is gained, so a person whose cells carry it still has two copies of every gene and is usually healthy. Such a person is a balanced carrier. The exceptions are a break that falls inside a gene, which disrupts it, and a break that places a gene beside a different promoter, which changes how much of it is transcribed; either can alter the phenotype although no DNA has gone missing.

The difficulty appears at meiosis. A carrier has one normal and one translocated copy of each of the two chromosomes involved, and each gamete receives one version of each. If the two pairs are treated as separating independently, four kinds of gamete are made in equal numbers: one with both normal chromosomes, one with both translocated chromosomes, which is balanced like the parent, and two that each carry one normal and one translocated chromosome. Those two are unbalanced: each carries one section twice and lacks another. An embryo formed from an unbalanced gamete has three copies of some genes and only one of others, and most such pregnancies end in miscarriage. In reality the four chromosomes pair up together as a cross-shaped group in prophase I, so segregation is not independent and the proportions vary from one translocation to another. A balanced translocation in one partner is among the causes looked for when a couple have had several miscarriages.

Describe the drawing

At the top, four chromosomes drawn as vertical bars, coloured cyan for material from chromosome A and coral for material from chromosome B. From left to right they are a normal A, an A whose tip has been replaced by the tip of B, a normal B, and a B whose tip has been replaced by the tip of A, so the carrier has every segment twice and is healthy. Below, four circles stand for the four kinds of gamete, each holding one version of A and one of B: a normal gamete, a balanced gamete with both swapped chromosomes, and two unbalanced gametes, one with the tip of A twice and no tip of B, the other the reverse.

Two translocations are met by name. In roughly one case of Down syndrome in twenty-five, the extra chromosome 21 material is attached to another chromosome, most often chromosome 14, rather than present as a separate third chromosome. One parent may be a balanced carrier, which raises the chance of a second affected child, so a karyotype of the child is used to tell the two kinds apart. And in chronic myeloid leukaemia the cancer cells carry a translocation between chromosomes 9 and 22 that joins part of a gene called BCR to part of the ABL gene. The fused gene codes for a permanently active enzyme that signals the cell to divide, so a proto-oncogene has become an oncogene by being moved, with no base substitution involved. That translocation arises in a single blood stem cell during a person's life and is not inherited; the drug imatinib, which blocks the fused enzyme, is the standard treatment.

Chromosome mutation

A change in the structure of a chromosome, or in the number of chromosomes, that can affect many genes at once.

Translocation (chromosome)

A chromosome mutation in which a section of one chromosome breaks off and joins a non-homologous chromosome.

Balanced translocation carrier

A person with a reciprocal translocation who has lost and gained no genes, is usually healthy, but can produce unbalanced gametes.

Mutations in coding sequences and in regulatory sequences

The genome comprises genes together with the sequences that determine when and how much each gene is transcribed: promoters, enhancers, the boundaries between introns and exons, and the genes coding for the transcription factors themselves. A change in any of those alters the amount of protein made, or when it is made, without altering its amino acid sequence.

Lactase persistence. Most adult humans stop making lactase after weaning, which is the ancestral condition. The variant that keeps it switched on in people of northern European descent is a single base change, and it is not in the lactase gene. It lies about fourteen thousand base pairs away, inside an intron of a neighbouring gene, in a sequence that acts as an enhancer for lactase. The lactase protein of a lactose-tolerant adult has the same amino acid sequence as anyone else's. The change is in a regulatory sequence.

β-thalassaemia. Many of the alleles causing it are not in the coding sequence of the β-globin gene at all. Some sit in the promoter and reduce transcription; others sit at an intron boundary, so the spliceosome cuts in the wrong place and the mature mRNA carries intron sequence. Either way the cell makes too little normal β-globin.

Tumours. A heavily methylated promoter can silence a gene whose sequence is intact. That case is treated in the next section.

Describe the drawing

A map along the top drawn to a base-pair scale shows two genes on one stretch of DNA: on the left a neighbouring gene whose long intron contains an amber block, the enhancer for lactase, and on the right the lactase gene itself with a cyan promoter and three coral exons separated by introns. A measured span between them is labelled fourteen thousand base pairs; the lactase gene is drawn shortened, since it really spans about forty-nine thousand. Beneath, three panels take the same event, one base swapped, in three places. In the first the chain of amino acids has one bead coloured differently, because one residue has changed. In the second the chain is unchanged bead for bead and two bars beside it show ten times as much protein being made. In the third, two messenger RNA bars are compared and the lower one is longer by exactly the length of the first intron, which the spliceosome has failed to remove. A band at the foot draws a healthy promoter and a methylated one on genes with identical coding sequence, the first transcribed normally and the second not transcribed at all.

Mutation

A change in the base sequence of DNA. Whether the protein changes depends on which base changes and where in the transcript it lies.

Silent mutation

A base substitution that produces a triplet coding for the same amino acid, so the primary structure of the polypeptide is unchanged.

Frame shift

The displacement of the reading frame caused by insertion or deletion of a number of bases that is not a multiple of three.

Control sequence

A region of DNA that is not translated but determines whether, when and how strongly a gene is transcribed.

Cystic fibrosis: one faulty channel, three organ systems

Pearson Edexcel Biology A builds its second topic round cystic fibrosis and asks how the mutation impairs the gaseous exchange, digestive and reproductive systems. The explanation runs through one protein, the CFTR protein (cystic fibrosis transmembrane conductance regulator), and one substance, mucus. Prerequisite: osmosis and the transport of ions across membranes.

CFTR is a channel protein in the cell-surface membrane of epithelial cells, on the side facing the lumen of the tube they line. When it is opened, which requires ATP, chloride ions leave the cell through it into the lumen. Sodium ions follow the chloride, attracted by its negative charge, passing between the cells. The extra ions lower the water potential of the liquid outside the cells, so water leaves the cells by osmosis. In the airways this keeps a layer of liquid deep enough for the cilia to beat in, with runny mucus on top of it. CFTR also restrains a sodium channel in the same membrane that takes sodium ions back into the cell.

In cystic fibrosis both alleles are faulty. With the commonest allele the protein is made but misfolds and is destroyed before it reaches the membrane, as described above; other alleles give a channel that reaches the membrane but rarely opens, or no protein at all. In every case too few chloride ions are secreted. Sodium ions are absorbed faster than normal, because the restraint on the sodium channel has gone, and water follows them into the cells by osmosis. Liquid is drawn off the epithelial surface, and the mucus loses water and becomes thick and sticky. Every tube lined by this kind of epithelium is affected in the same way; the three systems differ in what the blocked tube was for.

Describe the drawing

Two panels of airway lining side by side, each a row of three epithelial cells with a layer of liquid, cilia and mucus above them. On the left, with CFTR working, arrows show chloride ions, sodium ions and water all moving into the airway, the liquid layer is deep, the cilia stand upright, and the mucus on top is a thin band. On the right, in cystic fibrosis, chloride movement is marked as blocked, arrows show sodium ions and water moving into the cells, the liquid layer is almost gone, the cilia lie flattened, and the mucus is a thick, sticky band.

The gaseous exchange system. Mucus normally traps dust and bacteria, and the cilia sweep it up the airways to the throat. Dehydrated mucus is too thick for the cilia to move, and with the liquid layer gone they have no room to beat, so mucus collects in the bronchioles. Where it blocks a bronchiole completely, the alveoli beyond it are no longer ventilated, so the surface area available for gas exchange falls and less oxygen diffuses into the blood. Bacteria trapped in the stagnant mucus multiply, and repeated chest infections follow, Pseudomonas aeruginosa being a common cause. Each infection brings inflammation, and repeated inflammation leaves scar tissue that damages the lungs progressively. People with cystic fibrosis are short of breath, cough persistently, and do physiotherapy every day to clear the mucus.

The digestive system. The pancreas secretes amylase, lipase and proteases such as trypsin into the pancreatic duct, which carries them to the duodenum. In most people with cystic fibrosis the secretion in the duct is so thick that the duct becomes blocked, often before birth. The enzymes cannot reach the small intestine, so starch, fats and proteins are only partly digested and their products are not absorbed: the person struggles to gain weight however much they eat, and undigested fat passes out in the faeces. Trapped behind the blockage, the enzymes damage the pancreas itself, which becomes scarred and forms fluid-filled cysts, the fibrosis and cysts that gave the disease its name. Scarring can eventually reach the islets that secrete insulin, and diabetes is common in adults with the condition. Mucus lining the small intestine is also thicker, which slows absorption, and in some newborn babies the intestine is blocked outright. Most patients take capsules of pancreatic enzymes with every meal.

The reproductive system. In almost all men with cystic fibrosis the vas deferens, the tube that carries sperm from the testis towards the urethra, is blocked by thick secretion or is absent, having failed to develop before birth. Sperm are made normally but cannot leave, so the man is infertile, although sperm can be collected from the testis by a small operation and used in IVF. In women the mucus at the cervix is thicker than usual and sperm pass through it less easily, so fertility is reduced; many women with cystic fibrosis conceive without help.

Drugs called CFTR modulators now act on the protein itself. Some help a misfolded protein reach the membrane and others hold the channel open for longer, and combinations of the two are licensed for people with at least one copy of the commonest allele. They improve lung function substantially, but they cannot rebuild tissue that has already been destroyed.

CFTR protein

A chloride ion channel in the cell-surface membrane of epithelial cells; when it is faulty, too little chloride and water are secreted and mucus becomes thick and sticky.

Oncogenes and tumour suppressor genes

A tumour is a mass of cells produced by uncontrolled division. A benign tumour remains localised and does not metastasise. A malignant tumour invades surrounding tissue and may spread through blood or lymph to form secondary tumours. Growth rate, degree of differentiation and encapsulation can provide supporting evidence, but they are not the defining distinction. Spread, metastasis, is what makes a cancer difficult to treat surgically, because there is no single site to remove.

Two classes of gene are involved, and they are altered in opposite directions.

Describe the drawing

Two columns setting the two classes of cancer gene side by side. The left column follows a proto-oncogene, which normally makes a protein instructing the cell to divide when a growth signal arrives; a mutation leaves that protein permanently switched on, which the figure labels an accelerator jammed down, and one faulty allele suffices. The right column follows a tumour suppressor gene, which normally makes a protein that halts division or starts the cell's own death programme; heavy methylation of its promoter stops it being transcribed, which the figure labels a cut brake, and both alleles must be lost before it matters.

A proto-oncogene is a perfectly normal gene coding for a protein that stimulates cell division when a growth signal arrives: a growth factor, a receptor for one, or a protein that relays the signal inside the cell. A mutation can convert it into an oncogene: a version that is permanently active, or one that is present in too many copies, or one that has been moved next to a promoter that never switches off. The cell then receives a constant stimulus to divide whether or not any growth factor is present. One faulty allele is enough, because a permanently active protein does its damage regardless of the normal protein made by the other allele.

A tumour suppressor gene codes for a protein that does the opposite: it halts the cell cycle at a checkpoint, or triggers apoptosis in a cell whose DNA is damaged beyond repair. Loss of function removes that control, and in most cases both alleles must be lost, because one working copy still makes the protein. The two-copy requirement is the common case rather than a universal rule; for a few suppressor genes, loss of one copy already reduces the protein below the amount required. That accounts for inherited predisposition: someone born with one faulty BRCA1 allele does not have cancer, but every cell requires only one further event to lose the control, rather than the two events required in someone with two working alleles.

A third route involves no change to a base sequence. A tumour suppressor gene whose promoter becomes heavily methylated is transcribed less, or not at all, and the control is lost as it would be if the gene had been deleted, although the sequence is unaltered. This occurs in many tumours: the mismatch repair gene MLH1 is silenced this way in a substantial fraction of colorectal cancers, which explains why cancer is described as a disease of gene expression and not only a disease of mutation. Methylation can go wrong the other way too: hypomethylation of a proto-oncogene's promoter increases its transcription, so the growth-promoting protein is over-produced and the cell is stimulated to divide, as it would be by an oncogene.

Proto-oncogene

A normal gene whose product stimulates cell division in response to a growth signal.

Oncogene

A mutated or over-expressed proto-oncogene whose product stimulates division continuously.

Tumour suppressor gene

A gene whose product slows the cell cycle or triggers apoptosis; loss of both alleles removes that control.

Metastasis

The spread of cells from a malignant tumour through the blood or lymph to form secondary tumours.

Oestrogen has a role that should be stated precisely. Breast tissue is a target for oestrogen, and in a cell where a proto-oncogene lies downstream of an oestrogen response element, more oestrogen means more transcription of a gene that stimulates division. After the menopause the ovaries stop producing oestrogen but adipose tissue continues to, and higher concentrations of oestrogen in post-menopausal women are associated with a higher risk of breast cancer. That is an association supported by a plausible mechanism. It is not equivalent to the claim that oestrogen causes breast cancer in the way that loss of a tumour suppressor gene does. State which kind of claim an answer is making.

Stem cells, and the argument that surrounds them

Cancer is a failure of control over division. Stem cell medicine is an attempt to use controlled division deliberately, and the two sit in the same unit for that reason. You met the levels of potency earlier in the course; what matters here is where the cells come from and what they are being used for.

One use is genuinely routine: bone marrow transplantation replaces a patient's blood-forming stem cells after their own have been destroyed by leukaemia or its treatment, and has been standard care for decades. A second is licensed: sheets grown from a patient's own limbal stem cells restore the corneal surface after chemical burns. Beyond those, trials are running for macular degeneration, Parkinson's disease, type 1 diabetes and spinal cord injury, some with encouraging early results, and none of them is established treatment. When evaluating a stem cell claim, distinguish a treatment in clinical trial from one in routine use.

The ethical arguments are set out below rather than resolved. The disagreement turns on a question biology does not answer: what moral status a five-day-old human embryo has.

Those who oppose the use of embryonic stem cells generally hold that a human embryo has the full moral status of a person from fertilisation onwards, since that is when a genetically distinct human organism begins and no later point marks a non-arbitrary threshold. On that view, destroying an embryo to obtain its cells is the deliberate destruction of a human life, and the good that might be done with the cells does not license it. The Roman Catholic Church argues this position formally, and it is held by many people on secular grounds as well.

Those who support it generally hold that moral status develops gradually, and that an embryo of about a hundred cells with no nervous system, no capacity for experience and no individuality yet fixed does not have the interests that make killing a person wrong. On that view the suffering of identifiable patients with untreatable conditions carries more weight than the destruction of a blastocyst, particularly when the embryos used are surplus from IVF and would otherwise be discarded.

A middle position accepts the use of surplus IVF embryos while rejecting the creation of embryos specifically for research, on the ground that there is a moral difference between using something that will be destroyed anyway and bringing it into existence in order to destroy it. British law reflects a version of this compromise: research on human embryos requires a licence from the Human Fertilisation and Embryology Authority, and no embryo may be kept beyond fourteen days.

Induced pluripotent stem cells alter the terms of the argument without resolving it. They are made by treating an adult cell, usually a skin or blood cell, with a small set of transcription factors that switch previously silent genes back on, returning the cell to a pluripotent state. No embryo is involved and, because the cells come from the patient, tissue grown from them carries a much lower risk of rejection, reduced rather than abolished, since immune reactions to reprogrammed cells have been reported. That removes the objection raised against embryonic sources. Three limitations remain: reprogrammed cells can carry mutations acquired during the adult cell's life, their tendency to form tumours has to be controlled, and the fact that they can in principle be turned into gametes raises questions of its own.