Mutation

Mutation is the alteration of DNA sequence, whether it be in a small way by the alteration of a single base pair, or whether it be a gross event such as the gain or loss of an entire chromosome. It may be caused through the action of damaging chemicals, or radiation, or through the errors inherent in the DNA replication and repair reactions. One consequence may be genetic disease. However, although in the short term mutation may seem to be a BAD THING, in the long term it is essential to our existance. Without mutation there could be no change and without change life cannot evolve. If it had not been for mutation the world would still be covered in primeval slime!

In this course we are not going to consider the molecular events involved in mutation but instead will concentrate on the genetic consequences of mutation.

Somatic or germinal?

The first point to consider is where is the mutation occuring? Most of our cells are somatic cells and consequently most mutations are happening in somatic cells. New mutation is only of genetic consequence to the next generation if it occurs in a germ line cell so that it stands a chance of being inherited. That is not to say that somatic mutation is unimportant, cancer occurs as a direct consequence of somatic mutation and aging too may be caused at least in part by the accumulation of somatic mutations with time.

Different types of mutation occur at different frequencies:

type of mutation	mechanism	frequency per cell division
point mutation	1. mistakes in DNA replication 2. DNA damage by chemical mutagens (or by radiation) and misrepair	~10-10/basepair ~10-5/gene ~0.5/cell
submicroscopic deletion or insertion	1. unequal crossing over 2. misalignment during DNA replication 3. insertion of mobile element 4. DNA damage by chemical mutagens (or by radiation) and misrepair	included in the above
microscopically visible deletion, translocation or inversion	1. unequal crossing over 2. DNA damage by chemical mutagens (or by radiation) and misrepair	6 x 10-4
loss of a whole chromosome	missegregation at mitosis	1 in 100

Most gross mutations will impose a survival or growth rate disadvantage on the cell in which they occur and on the descendents of that cell which consequently will not contribute significantly to the body. The obvious exception is again cancer.

mosaicism

If a mutation such as a chromosome loss occurs early in development, the descendents of the cell may represent a significant fraction of the individual who, being composed of cells of more than one genotype is a genetic mosaic. (And see also X inactivation in lecture 4.) Mosaicism is not infrequent in for instance the cases of Turner's syndrome or Down's syndrome described later in this course. (Some of the links given in this paragraph are to lectures given to medical students.)

Most mutations are recessive

Why is this? Because most genes code for enzymes. If one gene is inactivated the reduction in the level of activity of the enzyme may not be as much as 50% because the level of transcription of the remaining gene can possibly be up regulated in response to any rise in the concentration of the substrate. Also, the protein itself may be subject to regulation (by phosphorylation for instance) so that its activity can be increased to compensate for any lack of numbers of molecules. In any case, if the enzyme does not control the rate limiting step in the biochemical pathway a reduction in the amount of product may not matter.

In the case of phenylketonuria (PKU) we can see that it is necessary to reduce the enzyme level to below 5% before any effect is apparent in the phenotype. This genetic disease is caused by mutations in the gene coding for the enzyme phenyalanine hydroxylase which converts the amino acid phenylalanine to tryosine. If an individual is homozygous for alleles which completely remove any enzyme activity, phenylalanine cannot be metabolised and it builds up in the circulation to a point where it begins to damage the developing brain. Newborn infants are routinely screened for this condition by the analysis of a tiny drop of blood from a heel prick (Guthrie test). This has revealed that there exist a few people with a condition known as benign hyperphenylalaninemia. These individuals have moderately elevated levels of phenylalanine in their serum. Their phenylalanine hydroxylase enzyme levels are about 5% of normal. Despite this they are apparently perfectly healthy and to not suffer from the brain abnormalities caused by the full blown disease.

What makes a mutation dominant?

• Haploinsufficiency.

  In this case, the amount of product from one gene is not enough to do a complete job. Perhaps the enzyme produced is responsible for a rate limiting step in a reaction pathway. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia leading to telangiectases and arteriovenous malformations of skin, mucosa, and viscera. Death by uncontrollable bleeding occasionally occurs. It is caused by mutation in the gene ENG, which codes for the protein endoglin, a transforming growth factor-beta (TGF-beta) binding protein. Perhaps the TGF-beta is unable to exert sufficient effect on cells when only half the normal amount of receptor is present.

• Dominant negative effect.

  The product of the defective gene interferes with the action of the normal allele. This is usually because the protein forms a multimer to be active. One defective component inserted into the multimer can destroy the activity of the whole complex. An example might be Osteogenesis imperfecta, see below

• Gain of function

  It is possible to imagine that by mutation a gene might gain a new activity, perhaps an enzyme active site might be altered so that it develops a specificty for a new substrate. That this must be so is self evident, how else could evolution occur? Examples in human genetics of genes with two such different alleles are rare and the only example which I can think of is the A and B alleles of the ABO blood group gene. There are many examples from human evolution however. Many genes have duplicated and subsequently the two duplicates have diverged in their substrate specificities. On chromosome 14 is a little cluster of three related genes, alpha -1-antitrypsin, (AAT), alpha -1-antichymotrypsin, (ACT), and a related gene which has diverged to such an extent that it is probably no longer functional. The structural relationship between AAT and ACT is very obvious and both are protease inhibitors but they now clearly serve slightly different roles because they have different activities against a range of proteases and they are under different regulation.

• Dominance at an organismal level but recessivity at a cellular level.

  Some of the best examples of this are to be found in the area of cancer genetics which will be taught later on. Some brief information can be found here. A typical example of such mutant gene would be a tumour suppressor gene such as retinoblastoma.

Point Mutation

'Point mutation' should strictly mean a single base pair alteration. However, in practice it is used more loosely to cover a variety of sins from genuine single base pair changes to small deletions and insertions (which I have however placed in the next section). A single base pair change may have no genetic consequences whatsoever but, on the other hand, it may cause a dominant lethal effect. The first alternative is far more likely. Why? Because 95% of DNA is non coding and a single base change occuring within it is unlikely to have any effect. In addition, because of the degeneracy of the genetic code, many mutations occuring within the third base position in a codon will have no consequence to the amino acid encoded.

The figure shows a short region of coding sequence with a variety of possible mutations.

amino acid substitution

A single amino acid change may be unimportant if it is conservative and occurs outside the active site of the protein. On the other hand it can have severe effects.

• The single substitution of valine for glutamic acid at position six of the beta-globin polypeptide chain gives rise to sickle cell disease in homozygotes because the modified chain has a tendency to crystalise at low O₂ concentrations.
• Collagen is a family of related structural proteins which are vital to the integrity of many tissues including skin and bones. The mature collagen molecule is comprised of three polypeptide chains wound in a triple helix. The chains first associate at their C termini and then twist together in a C to N direction. To be able to accomplish this collagen polypeptide chains have a special repeating structure of three amino acids, glycine - X - Y (X is usually proline and Y can be any of a wide range of aminoacids). A point mutation which by changing a single amino acid disrupts either the association of chains at their C termini or which prevents the triple helix formation may have very severe consequences. One mutant chain can disrupt a triple helix with two wild type chains effectively 'mopping up' functional monomers. The small amount of working collagen produced, not being an enzyme, cannot be upregulated. The consequence can be the dominant lethal condition osteogenesis imperfecta.

frame shift and nonsense

By nonsense is meant the premature insertion of a stop codon into the gene sequence. This might be by a single nucleotide change as in the figure above where the codon CAG which encodes glutamine (Q) has mutated to the stop codon TAG (UAG in the mRNA of course). Alternatively it can be as a consequence of a deletion or insertion of a number of nucleotides not divisible by three which shifts the reading frame and by chance will usually quickly lead to a stop codon. In the figure above, the same C nucleotide has been deleted in the last line leading to frameshift and a quick termination. The tuberous sclerosis gene TSC1 contains a direct repeat of four nucleotides, AAAGAAAG. Four independent mutations have been identified in which one repeat has been lost by deletion of AAAG. This leads to frameshift and premature chain termination.

triplet deletion

There is nothing particularly special about a triplet deletion which removes exactly one amino acid from the polypeptide (and which may change one amino acid at the mutation site). However, I include it because the most common mutation in cystic fibrosis is Delta F508 (i.e. deletion of amino acid number 508 (a phenylalanine, F)).

splice site

Frameshifts can also come about by mutations which interfere with mRNA splicing. The beginning and end of each intron in a gene are defined by conserved DNA sequences. If a nucleotide in one of the highly conserved positions is mutated then the site will no longer function with predictable consequences for the mature mRNA and the coded protein product.

There are many examples of such mutations, for instance, some beta thalassemia mutations in the beta globin gene are caused by splice junction mutations.

deletion and insertion

small i.e. less than 10kb

Deletions or expansions of a small number of nucleotides happen from time to time. Some deletions are entirely random but many are caused by misalignment of short repeats during DNA synthesis, so called replication slippage.

As much as 25% of the human genome is composed of repetitive DNA. Much of this is concentrated at chromosome centromes and in heterochromatic regions but a great deal of it is in the form of interspersed repetitive DNA. There are several different types of this but the two principal types are alu elements and L1 repeat elements. These interspersed repeats are very important from an evolutionary point of view. Working elements can transpose, via an RNA intermediate. New copies of the element can be inserted at random into the host genome. Sometimes, if the insertion takes place within an exon or within a control region, this can cause a mutation. Such fresh insertions are very rare events. The presence of numerous repeats does however have other effects. If two alu elements are close together in the genome they may misalign at meiosis and if a recombination event takes place the consequence will be a ganmete with either one fewer or one more copy of the element and the intervening DNA sequence.

Large but still submicroscopic i.e. less than 1Mb

Almost all cases of X linked ichthyosis are brought about by total deletion of the gene STS, coding for steroid sulphatase. This is caused by a mutation which seems to be recurrent. The STS gene is flanked by two copies of a repetitve element. It is thought that sometimes in meiosis these two copies, on the same chromatid, align and recombine leading to excision of the DNA between them as in the figure below.

Sometimes deletions can cover more than one gene. When this occurs we have an example of a contiguous gene syndrome. For instance, some patients with Tuberous sclerosis also suffer from polycystic kidney disease. The two loci TSC2 and PKD are adjacent on chromosome 16 and can be simultaneously deleted.

Cytogenetic

Some deletions become so large that they are visible on well stretched metaphase chromosomes. When enough of the genome is deleted to be visible in this way, the symptoms are usually very severe because a large number (probably more than a hundred) genes will have been lost. Contiguous gene syndromes will become likely. More on this later.

Trinucleotide expansion

The commonest inherited cause of mental retardation is a syndrome originally known as Martin-Bell syndrome. Patients are most usually male, have a characteristic elongated face and numerous other abnormalities including greatly enlarged testes. The pattern of inheritance of this disease was, at first, puzzling. It usually behaved as an X linked recessive condition but sometimes manifested itself in females and occasionally nonaffected transmitting males were found. In 1969 it was discovered that if cells from patients were cultured in medium deficient in folic acid their X chromosomes often displayed a secondary constriction near the end of the long arm. The name of the syndrome was changed to the fragile X syndrome. The puzzling genetics remained unclear. Eventually the mutation was tracked down to a trinucleotide expansion in the gene now named FMR1 (Fragile site with Mental Retardation) at the site of the secondary constriction. As in the case of myotonic dystrophy symptomless premutations could occur (and were the cause of the transmitting males). Only when the premutation chromosomes were transmitted through females did expansion to the full mutant allele and phenotype occur. A number of diseases have now been ascribed to trinucleotide expansions. These include Huntington's disease.

Recommended reading

The topics include all aspects of mutation

Reading:

• Mange and Mange Chapter 7 (pp155 - 158), 12 (pp270 - 282)
• Lewis Chapter 10
• Mueller and Young Chapter 2 (pp16 - 20), and, as further reading, chapter 9
• Thompson McInnes and Willard Chapter 6 (Chapters 11 and 12 make interesting further reading)
• Connor and Ferguson-Smith Chapter 2 (pp17 - 23)

SAQs

1. The following short sentence represents a gene:
   

   WET WET WET ARE NOT ALL BAD

   What types of mutations are the following?

   1. WEE WET WET ARE NOT ALL BAD

   2. WET WET WET ARE COT ALL BAD

   3. WEW ETW ETA REN OTA LLB AD

   4. WET WET WET ART TEN TAL LBA D

   5. WET WET ARE NOT ALL BAD

   6. WET WET WEG OOD MOR NIN GTA REN OTA LLB AD

   7. WET WET WET WET WET WET ARE NOT ALL BAD

2. Full colour vision requires that the retinal cone cells be of three types differing in which of three opsin molecules they produce. One of the three opsin genes, that coding for a blue sensitive protein, is autosomal but the other two genes (red sensitive and green sensitive respectively) are adjacent to each other on the X chromosome. What are the consequences of this arrangement of genes for the mutation frequency of the X linked genes?