The different types of mutation: The different types of mutation


Slide2: Mutations can be DOMINANT or RECESSIVE For recessive alleles, the mutant phenotype is only expressed in the absence of the wild-type allele. Genotype Phenotype m/m M m/+ Wild-type For dominant mutations one can see the mutant phenotype even in the presence of the wild-type allele. Genotype Phenotype m/m M m/+ M


Slide3: How can a mutation be dominant? (i) By increasing the normal activity of the wild-type allele (HYPERMORPHIC). (iv) If one copy of the gene provides insufficient material (HAPLOINSUFFICIENT). (iii) By conferring a new activity on the protein (NEOMORPHIC). (ii) By interferring with the activity of the wild-type allele (DOMINANT NEGATIVE).


HYPERMORPHS: HYPERMORPHS Hypermorphic => Increase in nomal wild-type activity. An example of a hypermorphic mutation is a change in codon 12 of the Ras gene. This mutation is found in a significant fraction of human tumours. Ras is part of a signal transduction pathway that is activated by the growth factor EGF. The normal function of Ras is to activate a serine threonine kinase called Raf. Ras can exist in either of two states: one bound by the guanine nucleotide GDP (inactive), and one bound by GTP (active). Generation of the active, GTP-bound form is stimulated by guanine nucleotide exchange factor (GNEF). The GDP-bound form is generated from the GTP-bound form by hydrolysis of GTP to GDP. Wild-type Ras has intrinsic GTPase activity. This intrinsic activity is stimulated by GTPase-activating protein,GAP.


Slide5: HYPERmorphic mutations in Ras, lock the protein in the GTP-bound active state by interferring with the ability of GAP to stimulate GTP hydrolysis. The net effect is to increase the amount of Ras activity in the cell. Some cells with constitutively active Ras divide uncontrollably. Many human tumours (approx. 30%) contain cells with hypermorphic Ras mutations.


DOMINANT NEGATIVE: DOMINANT NEGATIVE Dominant negative mutations have two effects (i) they cause the mutant protein itself to have reduced or no activity. (ii) In heterozygous individuals, the mutant protein also blocks the function of the protein encoded by the wild-type allele. dn/+ = mutant. Dominant negative mutations are known in Ras. Mutant protein binds to GNEF and inactivates it. No GNEF is then present in the cell to activate wild-type Ras. Dominant negative mutations can exert their effects on the wild-type protein in either of two ways: (i) by directly binding to, and inactivating the wild-type protein. (ii) by binding to and inactivating a second protein that is required for the wild-type protein’s function.


Slide7: Dominant negative p53 mutations are common in human tumours. p53 is a TUMOUR SUPPRESSOR. => absence of p53 activity contributes to the cancer phenotype. p53 is a transcriptional regulator. The protein functions as a tetromere. Mutant p53 protein is incorporated into the tetromere and disrupts its function. Dominant negative mutations are also called ANTIMORPHIC mutations.


NEOMORPHIC: NEOMORPHIC Neomorphic mutations cause the mutant protein to acquire a new activity not associated with the wild-type protein. For example, the protein could gain the ability to bind to a protein to which it does not normally bind. Or cleave a substrate upon which it does not normally act. Or form insoluable multimers that the wild-type protein does not give rise to. Or a host of other possibilitites...


HAPLOINSUFFICIENT: HAPLOINSUFFICIENT The majority of proteins in the cell are present at considerably higher concentrations than they are needed. Thus halving the amount of protein in the cell has no deleterious consequences (m/+ = WT). Some proteins, however, are present at limiting concentrations. Reducing the amount of such a protein by half causes there to be too little in the cell. Such genes are said to be HAPLOINSUFFICIENT. Half is not enough. For these genes, mutations that reduce or eliminate activity are dominant. m/+ = mutant


HYPOMORPHS and AMORPHS: HYPOMORPHS and AMORPHS Hypomorphic mutations REDUCE but do not eliminate gene activity. (Also called REDUCTION-OF-FUNCTION.) Amorphic mutations completely eliminate gene activity. (Also called NULL mutations.) Because it is much easier to disrupt a protein’s function than it is to activate it (or give it a new function), most mutations are either hypomorphic or amorphic.


Slide11: In human genetics one starts typically with a genetic disease and tries to find out the cause by isolating the mutated gene or genes. In genetic studies with model organisms, one starts with a process one is interested in and then deliberately tries to find mutations that disrupt the process. This is so-called FORWARD genetics. ”In the same way that a novice automechanic starts to learn about the workings of an internal combustion engine by examining the effect of taking out let us say a spark plug, a geneticist ’tinkers’ with living systems by remving things that are vital for its proper working.” David Suzuki. ”Don’t it always seem to go that you don’t know what you’ve got ’till it’s gone.” Joni Mitchell.


Slide12: Suppose one is studying a mutation that causes a phenotype, P. (It could either be a mutation associated with a human genetic disease or a mutation in a genetic model organism.) The mutation could either be in a gene whose normal function is in the proces affected by the mutation. Or the mutation could be neomorphic. In this case the affected protein does not normally funcion in the process in question. The mutation confers on the mutant protein a new activity: the ability to disrupt the process one is studying. Because neomorphic mutations exist, one cannot necessarily conclude that a mutation that disrupts a process, defines a gene that normally functions in that process.


Conclusion:: Conclusion: In order to know whether a certain gene really is involved in the process one is studying, one needs to know whether or not the mutation is neomorphic. If one discovers that the mutation REDUCES or ELIMINATES gene activity, then one can be sure that the wild-type protein IS REQUIRED FOR the process one is studying.


Slide14: Imagine yourself as the geneticist car mechanic who is trying to figure out how cars change direction. When he removed the steering wheel, the cars were no longer able to make turns. Therefore, steering wheels ARE REQUIRED FOR cars to change direction. Q. What would have happened if he had removed the bolt that fixed the steering wheel to the steering column? A. The steering wheel would not have worked and the car would not have been able to change direction. Thus the bolt is also REQUIRED FOR cars to change direction. The geneticist is therefore left with the problem: How does he determine which is more important, the steering wheel or the bolt? In genetics with real cells, how does a geneticist know that the mutation he or she has isolated defines a gene that is intimately involved in the process he is studying i.e. a steering wheel rather than a bolt. A. One way is to isolate HYPERMORPHIC mutations.


An example:: An example: Paul Nurse, winner of the Nobel prize for medicine 2001, received his prize for his work on elucidating the mechnisms that control the rate at which eukaryotic cells divide. In order to identify the proteins that control cell division in the yeast cell Schizosaccaromyces pombe he first carried out a GENETIC SCREEN for mutations that disrupted the ability of cells to divide. The cells got bigger and bigger but failed to divide. He isolated many mutants that he named cdc1, cdc2, cdc3… He then screened for mutations that had the opposite effect: mutations that caused cells to divide too soon. (They divided when they were still quite small.) He isolated a few mutants and called them wee1, wee2, wee3 … He found that cdc2 and wee1 were one and the same gene. Loss-of-function mutations in this gene cause cells to be unable to divide whereas hypermorphic mutations cause cells to divide too soon. He knew from these observations that cdc2/wee1 was the master regulator of cell division.


Another example:: Another example: Dr. H. Robert Horvitz, winner of the Nobel prize for medicine 2002, received his prize for elucidating the mechanisms controlling programmed cell death. Many cells in multicellular organisms are born only to die a short time later (or when they are no longer needed). Cells undergoing programmed cell death (also called apoptosis) die in a stereotypic manner involving distinct morphological changes. By carrying ou genetic screens with the nematode C. elegans (in which a strictly defined number of cells die), Horvitz identified a number of genes that regulated cell death. He discovered that HYPERMORPHIC mutations in ced-9 blocked cell death whereas LOSS-OF-FUNCTION mutations in the same gene caused all cells to die. Thus ced-9 is a master regulator of cell death.


Conclusion: Conclusion If you want to win a Nobel prize, you should do genetics!


Slide18: How does one distinguish between the different types of mutation, hypermporphic, amorphic, hypomorphic, antimorphic and amorphic? A. By gene dosage studies. To begin with: Hypermorphic and neomorphic mutations are usually dominant. Genotype Phenotype m/+ Mutant Hypomorphic and amorphic mutations are usually recessive. Genotype Phenotype m/+ Wild type


Expressivity and penetrance: Expressivity and penetrance Sometimes not all the individuals carrying a mutation show the mutant phenotype. e.g. Genotype Phenotype m/+ 60% M, 40% WT Such mutations are said to be INCOMPLETELY PENETRANT. Both recessive and dominant mutations can show incomplete penetrance. Genotype Phenotype m’/m’ 70% M, 30% WT


Slide20: Some mutations cause variable EXPRESSIVITY. Different individuals carrying the same mutation are affected to different degrees. Imagine, for example, that a mutation causes defects in the central nervous system. In some patients the mutation might cause difficulty walking, in others complete paralysis. Both recessive and dominant mutations can cause variable expressivity.


Slide21: One can determine the nature of a mutation by examining the effects on penetrance and expresivity of adding additional wild-type alleles. Antimorphic/+/+ are less mutant than Antimorphic/+ Hypermorphic/+/+ is more mutant than Hypermorphic/+ Neomorphic/+/+ has the same phenotype as neomorphic/+


Slide22: Or taking away wild-type copies of the gene: Hypomorphic/Df > hypomorphic/hypomorphic Amorphic/Df = amorphic/amorphic If +/Df is mutant then at least one gene under the deficiency is haploinsufficient. In C. elegans and Drosophila, extra copies of the wild-type gene are added by using chromosmal duplications. Wild-type copies are removed by using chromosmal deficiencies.