1er curso de actualización en biología molecular christian a. garcía sepúlveda md phd facultad...

1er Curso de Actualización en Biología Molecular

Christian A. García Sepúlveda MD PhD

Facultad de MedicinaUniversidad Autónoma de San Luis Potosí

Sesión #6

Mutaciones

2

Sesión #6 Mutaciones

Introducción

Mutation is a heritable change in the genome. There are many different classes of change.

•A mutation is a change in the DNA from its original form (mutatio = change, alteration in Latin)

•When just one base is changed in the DNA, it is considered a mutation. It would also create a new allele for the gene. Not all mutations are harmful.

3


Introducción

• A. Chromosomal Mutations

– Aneuploidy or a loss or gain of a whole chromosome: occurs when mitosis or meiosis doesn’t function correctly and causes a cell to have 46 +/or- 1 or 2 chromosomes e.g. X0 = Turner’s syndrome

– Polyploidy or a loss or gain of a whole set of chromosomes: instead of having 46 chromosomes, an additional 23 chromosomes are added or 23 are lost! (not found in humans but can occur in plants!!)

– Loss of a part of an arm of a chromosome = translocation. This occurs when the arm of one chromosome is attached to a different chromosome. (Could be reciprocal where both arms are attached to the other recipient chromosome)

– Inversions: where a portion of a chromosome rearranges the order of the DNA inside the arm

– Deletions: a large piece of DNA is taken out of the chromosome

– Duplication: a large segment of DNA is copied and inserted beside its original sequence.

– Insertion: a large piece of DNA coming from one chromosome and put into another

4


Introducción

– In a chromosome mutation, the large scale structure of a chromosome or the overall number of chormosomes are affected

– Deletions, Duplications, Inversions, Translocations are examples of structural changes.

– Deletions - loss of chromosome segment due to breakage and lack of repair.

– In heterozygous form may be deleterious due to dosage problems or the "unmasking of a recessive".

– If homozygous, it will be lethal (assuming the deleted region is important.)

5


Introducción

• B. Point Mutations– i) Insertion: 1 or 2 bases are added to

DNA at one place (not in multiples of 3 – why is that?)

• These mutations can cause serious effects if it occurs within a gene as the triplet sequence will be disrupted

– ii) Deletion: 1 or 2 bases are added to DNA at one place (not in multiples of 3 – why is that?)

• These mutations can cause serious effects as well if it occurs within a gene as the triplet sequence is disrupted

– Iii) Substitution: of 1 base for another base e.g. A T or G C

• These mutations may or may not be harmful depending on where the change in the gene occurs and the effect of this change on the resulting amino acid that it is changed to.

i)TAC GCT AGG ATG TAC GGC TAG GAT G

ii) TAC GCT AGG ATG

TAC CTA GGA TG

iii)TAC GCT AGG ATG

TAC ACT AGG ATG

6


Introducción

Base substitutions

•All typesCommon in coding and non-coding DNA

•Transition and TransversionTransitions are more common that transversion

•Synonymous and non-synonymousSynonymous are more common

•Gene conversion-like eventsRare except at certain tandemly repeated loci or clustered repeats

7


Introducción

Mutation is usually associated with deleterious effects on phenotype.

Changes at the DNA and protein level that are not obviously deleterious to phenotype are refered to as polymorphisms.

Allelic variation is referred to as polymorphism. In fact the amount of variation in the human genome is quite high.

1 in 250 to 1 in 1000 bases are different between alleles.

Simple mutations

Mutations can occur due to errors in replication and repair of DNA.

Replication errors occur at frequency of 10-9 to 10-11.

Adult human needs 1017 cell divisions to produce 1014 cells each new cell needs to incorporate 6 x 109 new nucleotides complete fidelity needs 6 x 1026. The average coding region for a gene is 1.7 kb this means that the average frequency of mutation per gene per cell division is 1.7 x 10-6 to 1.7 x 10-8.


Introducción

9


Clasificación

Insertions

One or a few nucleotidesVery common in non-coding DNA but rare in coding DNA where they produce frameshifts.

Triplet expansion Rare but contribute to several disorders.

Large insertionsRare but get large scale tandem duplications and transposible elements.

10


Clasificación

Deletions

One or a few nucleotides

Very common in non-coding DNA but rate in coding regions as they produce frameshifts

Larger deletions

Rare but get large scale tandem repeats deletions and transposible elements

Chromosome abnormalities

NumericalRare as constitutional mutations but can often be pathogenic

StructuralMore common as somatic mutations and often associated with tumor cells.


Clasificación

The type of cell that a mutation occurs is also important.

If a mutation occurs in a somatic cell CANCER may develop.

However if the mutation occurs in a germ cell it can be passed on to the next generation.

Mutations occur at a very low frequency. So most of the variation that is found in the genome of a one individual is due to inherited changes and not de novo mutations.


Clasificación

TRANSITION mutations

Substitution of a purine (A or G) for another purine OR substitution of a pyrimidine (C or T) for another pyrimidine.

TRANSVERSION mutations

Substitution of a purine (A or G) for a pyrimidine of vice versa.

Theoretically transversion should be 2 x more frequent than transitions.

Transitions occur 1.4 times more frequently than transversions.

Due to C →T transition due to 5-m C deamination.

CpG has a mutation rate 8.5 times higher than any other dinucleotide.

A ⇆ C⇅ ⇅ G ⇆ T


Transiciones y transversiones

Mutations are generated randomly in DNA, however only 3% of the DNA codes for products. Mutations in coding regions are classified as:

Silent (synonymous): do not change the product, therefore are neutral.

Nonsynonymous: mutations that result in a change in the protein or functional RNA. Most nonsynonymous mutations are deleterious, causing problems in the products produced. This can result in disease or lethality.

Nonsense mutations, Missense mutations

The location of base substitutions in coding DNA is non random. Why? Due to selection pressure on the base change.

Natural selection: is the process by which mutational changes are fixed or eliminated from the population.


Sinónimas y no-sinónimas

Mutations in Coding DNA

If one analyzes the mutations in coding DNA we see that they are non random. Due to the way the genetic code is set up there is redundancy that makes certain codons less susceptible changes in the protein product. See below…



Rate of nucleotide substitution varies depending on the position in the genome as well as in genes.



Rates of Synonymous and non synonymous mutations are not the same genome wide. It depend on the selective pressure for that specific gene!



Mutation rate is higher in males.

It has been observed that many mutations are derived paternally. In some cases 4.5 to 8.5 times as high.

This is due to the higher number of cell divisions that are necessary for the production of sperm.


Diferencias sexuales de mutaciones

Examples of genes that show an increase mutation rate from paternal sources.


Diferencias sexuales de mutaciones

Slipped strandMisspairingCausesDeletions andInsertions During DNA Replication.


Inserciones y deleciones

Other mechanisms for tandem repeat expansion or contraction.


DNA repetitivo y mutaciones

Unequal crossover can lead to homogenization.

DNA sequences can be homogenized by unequal crossing over.

Tandem duplication can occur by unequal sister chromatid exchange



Gene conversion is the non reciprocal exchange between alleles. It can occur between alleles on homologous chromosomes or in rare cases between two different related loci.


Conversión génica


Implicaciones funcionales

Effects of mutations on Proteins

• Positive – causes the protein to be have an even better function/ does its job better (this will allow for natural selection and evolution)

• Negative – causes the protein to have little or no function OR disrupts another protein’s function e.g. Sickle cell anemia

• Neutral – causes the protein to have no significant change in function (many are in this category such that we never see disease from these mutations)



Effects of mutations on Proteins

A. Normal protein B. Neutral mutation

C. Negative mutation D. Positive mutation

2 active sites now, more effective protein

No active sites

No change in the active site

Mutation can affect function in a number of ways

Examples of mutations that affect gene function --->



Mutation can affect function in a number of ways



Mutations that affect splicing Mutations can affect each process involved in the flow of information from DNA to proteins.Here we see that mutants can affect the process of splicing add or deleting information into the mature mRNA.



Example of a mutation that affect splicing LGMD2A



Creation of a cryptic splice site.



Tandem repeats are found through out the genome.

Give rise to various types of mutationdeletions and duplications and some inversions as well as insertions (retroviral)

Repeated elements

Deletions due to short tandem repeats.



Triplet repeat expansion

Uninterrupted triplets are more prone to expansion that those interrupted by other sequences

Triplet types



VNTRsVariable number tandem repeats are important in disease. They can contract or expand, but expansion is usually what is seen in certain disease states. Slipped strand mispairing is one of the mechanisms that can lead to changes in VNTR numbers.



Gene conversion

25% mutations Involve deletions

The rest are geneConversions.

Deletions or Duplications can be due to recombination or gene conversion


Conversión génica

Mutations in the 21-hydroxylase gene suggest gene conversionas the mechanism of mutagenesis.


Conversión génica

Direct repeats have been shown to be involved in deletionsDue to recombination in mutation in the human mitochondrial DNA. Due to slipped strand misspairing



Recombination is hypothesized to have produced thisMutation in the factor VIII gene that was identified in one patient.


Recombinación y mutaciones

• Variation is due to multiple mechanisms– Single bases, -> DNA replication, DNA damage…– Multiple bases,-> DNA replication, Recombination,

gene conversion, DNA damage…

• Changes can be Synonymous or non-synonymous for their effect in proteins. Selection acts at this level.

• Males have higher mutation rate due to more cell divisions; DNA replication…


Conclusiones

• Repeated elements expand and contract by slipstrand misspairing or unequal crossing over.

• Mutations can occur in many different parts of a gene and give rise to different severities. Mutations affecting splicing also affect the protein.

• Recombination and gene conversion can give rise to changes in the genetic material, some that are deleterious.


Conclusiones


Single Nucleotide Polymorphisms (SNPs)

Single nucleotide polymorphisms or SNPs are DNA sequence variations that occur when a single nucleotide (A,T,C or G) in the genome sequence is alteredFor a variation to be considered a SNP, it must occur in at least 1% of the populationIf the frequency is less than 1% (although this is somewhat arbitrary) then this variation is called a mutationSNPs are classified in three different ways…



Homozygous WT Heterozygous Homozygous Var.

Zygosity and SNPs



SNPs account for about 90% of all human genetic variation and are believed to occur every 100 to 300 bases along the 3-billion-base human genome

Approximately 5 million of the ~10 million human SNPs have been catalogued

SNPs may occur in exons, introns (non coding regions between exons) and intergenic regions (regions between genes)

SNPs may lead to coding or amino acid sequence changes (non-synonymous) or they may leave the sequence unchanged (synonymous)



Hardy Weinberg Equilibrium

Synonymous vs. Non-Synonymous SNPs



Measuring SNPs

– Classical sequencing (homozygotes)

– Chromatogram analysis (heterozygotes)

– Denaturing HPLC

– Rolling Circle Amplification

– Antibody-based detection

– Enzyme- or cleavage-based detection

– Mass spectrometry

– SNP chips or microarrays




True SNPs should follow Hardy Weinberg Equilibrium in that

The choice of a mate is not influenced by his/her genotype at the locus/gene (random mating or panmixia)

The locus/gene/SNP does not affect the chance of mating at all, either by altering fertility or decreasing survival to reproductive age



Deviations from HWE

– Marital assortment: "like marrying like"

– Inbreeding

– Population stratification: multiple subgroups are present within the population, each of which mates only within its own group (homogamy)

– Decreased viability of a particular genotype (hemophilia)

In Vitro Mutagenesis

Site specific mutagenesisZoller and Smith 1987 Methods of Enzymology 154,329-350Won Smith the Nobel prize in 1993 for Chemistry

M13 single stranded phase DNA annealed to a mutagenicPrimer and then in vitro synthesize the remaining DNA

The DNA template is obtained from plasmids or phage grown in a dut ung mutant of E. coli. The dut gene encodes dUTPase which normally degrades dUTP. An elevated concentration of dUTP accumulates in dut strains, resulting in incorporation of U in place T at some positions during DNA replication. The ung gene encodes uracil N-glycosylase which normally removes U from DNA. Thus, in the double mutant U is occasionally incorporated into DNA and this error is not repaired. Because U has the same base pairing properties and the same coding properties as T, incorporation of U into DNA in place of T is not mutagenic.

In Vitro Mutagenesis dut ung

The mutS gene encodes an essential componantof the methyl-directed mismatch repair system. Thus, a mutS mutation in the recipient prevents repair of the mutant base to the wild-type sequence, so that after DNA replication about 50% of the plasmids will be mutant and 50% will be wild-type. An very clever variation of the mutS approach was developed by scientists at Promega and is marketed as the "Altered Sites Kit". A figure of this approach is shown below and a more detailed description of this technique can be found on the Promega WWW site.

In Vitro Mutagenesis mutS

http://www.promega.com/tbs/tm001/tm001.html

In Vitro Mutagenesis PCR

1er curso de actualización en biología molecular christian a. garcía sepúlveda md phd facultad...

Documents