It is obvious to us all that we all differ from one another, and that many of these differences ‘run in families’. Apart from identical twins people can readily be distinguished, from their facial features and many other attributes. This high level of individuality is reflected in our DNA. Both non-coding and coding DNA show a great deal of person to person variation. The existence of variation at a molecular and biochemical level has been known for nearly 100 years, that is long before the first human DNA sequences were read. Some of the early biochemical traits to be identified took more than 50 years to be elucidated at the level of the gene. In contrast more recently the gene defect in many genetic diseases has been elucidated by sequencing of DNA with no knowledge at all of the function of the protein product.
Genetic variation in functional regions of the genome can fall into several different categories with respect to its effect on the individual and the frequency of the allele in the population.
A single amino-acid substitution can have very severe effects but it may be unimportant if it is outside critical regions of functional importance.
These are rare disorders in which an enzyme is deficient - which causes a block or 'error' in a metabolic pathway. They are usually recessive. Enzymes are catalysts - so that the half levels present in heterozygotes are sufficient, and these individuals are usually completely unaffected. The first inborn errors were described early in the 20th century by Sir Archibald Garrod. Garrod's concept of these disorders came mainly from his studies on the rare disorder Alkaptonuria. This is a relatively benign disorder but often diagnosed in infancy because of brown discoloration of the baby's nappy. The disorder is characterised by the massive urinary excretion of the substance homogentisic acid which is not normally found in the urine. Although the affected individuals are usually quite healthy - in later life they are particularly prone to develop a form of arthritis known as ochronosis - because of deposition of a substance derived from homogentisic acid.
Garrod observed that frequently more than one sibling in a family was affected and that often the parents were related (the marriages were consanguinous) and learned that these observations could be readily explained if the defect was inherited as a recessive condition in terms of the recently rediscovered laws of Mendel. Garrod was able to predict the enzyme deficiency. As recently as 1997 the gene encoding homogentisic acid oxidase was cloned and the first mutations identified. From his study of alkaptonuria Garrod developed the concept that certain diseases of lifelong duration arise because an enzyme governing a single metabolic step is reduced in activity or missing. Many inborn errors of metabolism are now known. One of the most common is phenylketonuria which gives rise to a clinical disorder which includes severe mental retardation. Phenylketonuria is due to deficiency of the enzyme phenylalanine hydroxylase which converts phenylalanine to tryrosine (see the pathway above). An effective treatment of this condition is to withhold phenylalanine from the diet.
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 crystallise at low O2 concentrations. The amino-acid substitution causes changes in electrophoretic mobility so that HbS and HbA can be separated by electrophoresis. The sickle cell trait in heterozygous carriers confers resistence to malaria.
A polymorphismis defined as the occurrence in a population of two or more common alleles. A locus is regarded polymorphic if the frequency of the rarest allele is more than 0.01 - ie if the heterozyote frequency is 2% or more.
The existence of human blood groups has been known since the beginning of the 20th century. Landsteiner showed that when suspensions of red blood cells obtained from different people are mixed with blood serum obtained from other people that the red cells often agglutinated and a clear cut pattern of differences in reaction was observed. By working out the patterns of the agglutinations he first defined the ABO blood groups. Others (Epstein and Ottenberg) showed that the ABO blood group was inherited as a Mendelian trait.
This was the first example of a human polymorphism (other than eye colour) inherited as a single Mendelian trait.
|Genotype||Phenotype||red cell antigens||serum antibodies|
|AB||AB||A and B||neither|
|OO||O||neither||anti-A and anti-B|
The ABO gene codes for a glycosyltransferase which adds a sugar residue to a carbohydrate structure known as the H antigen on the surface of red blood cells. The A allele codes for an enzyme which adds N-acetylgalactosamine, whereas the enzyme coded for by the B allele has two amino acid differences which alter is specificity and it adds D-galactose, forming the A and B antigens respectively. The O allele has a frameshift muation in the gene and thus produces a truncated and inactive product which cannot modify H. A phenotype people have natural antibodies to B antigen in their serum and vice versa. O phenotype individuals have antibodies directed against both A and B. AB individuals have no antibodies against either A or B antigens.
It has been known for a long time that common genetically determined variations which are not disease-causing in themselves can lead to disease in response to environmental exposure to substances to which other individuals do not respond adversely.
The first examples of this were discovered in the early 1950s. It seems very possible there will be a rush of new examples in the next few years as we enter the ‘post-genome’ era.
Inherited variations of serum cholinesterase came to light following the introduction and wide-spread use of the drug suxamethonium (succinyl dicholine) as a muscle relaxant in surgery. The effects of this drug are quite short lived in most people - but 1 in 2000 are unusually sensitive because the drug takes longer to degrade. It is usually metabolised by serum cholinesterase which hydrolyses the molecule as indicated. Some individuals carry a variant serum cholinesterase with reduced activity and altered kinetic properties.
Also in the 1950s it was noted that in some populations a significant number of individuals respond adversely to the anti-malarial drug primaquine or that some individuals are sensitive to fava (or broad) beans. These agents cause acute haemolysis in these people.
The sensitivities are due to deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD). Males are more commonly affected than females. This is because the gene encoding G6PD is X-linked.
Measurement of the level of G6PD activity in different individuals shows that the distribution of activities is bimodal in males in these populations, and broad and almost unimodal in females. The levels of activity in females are not twice the level that are seen in males, because only one of the X chromosomes is active in each cell.
With the advent of the technique of gel electrophoresis, it was possible to assess the frequency of normal genetically determined polymorphism of proteins. This is because electrophoresis can readily separate proteins which differ in charge. Because the allelic forms of the protein can be separated, codominant inheritance can be observed.
Electrophoresis is the separation of charged particles in an electric field - shown below:
The charge on protein depends on the ionisation of the R groups of the amino-acids.
‘basic’ proteins i.e. those rich in lysine, arginine, histidine are cathodal
‘acid’ proteins i.e. those rich in aspartic acid and glutamic acid are anodal.
A wide variety of changes can result from mutations in DNA. These include single nucleotide changes as well as large deletions, insertions and rearrangements. Mutations will be covered in more detail in a later lecture.
Here we will consider just one example - mutations in the coding region of the gene which lead to an amino-acid substitition through alteration of a codon. For example:
Arginine is positively charged at neutral pH because of the ionisation of the amino groups shown in bold.