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MUTATION AND POLYMORFISM
Genetics and genomics for ED students
20.02.2015.
Genetic variability
- is increased by – mutation
– sexual reproduction
meiosis (generation of gametes)
- homologous recombination (crossing over)
- independent assortment of homologous
chromosomes
fertilisation
- significance
DNA
Mutation
causing any
change)
DNA variants, alleles
(any coding or non-coding sequence)
Genetic (DNA) polymorphism
Significance of mutation (for all species)
Without mutation, evolution would not be
possible. This is because mutations
provide the "raw material" upon which the
mechanisms of natural selection can act.
Regarding the variants….
• normal or wild variant (allele) is the most frequent in a
population
• polymorphism (or polymorphic) is the variant (allele) if its
frequency is › 1 % in the population
(formerly:having no effect on phenotype)
• mutation (or mutant) is the variant (allele) if its
frequency is ‹ 1 % in the population
(formerly: disease causing, it has a
negative connotation)
Long way from mutation to
polymorphism
Appearance of
new variant
by mutation
Survival of rare allele
Increase in allele frequency
after population expand
New allele is fixed
in population as novel polymorphism
Classification of mutation types
• by the cause
• by the site
• by the function
• by the fitness
• by the size
By the cause mutations may be
• Spontaneous
• Induced
Spontaneous mutation
- Spontaneous chemical reactions in bases
• Tautomerization
• Depurination
• Deamination
- Errors in DNA related processes
• Replication
• Recombination
• Repair
E.g. Tautomers of adenine
Frequent
rare
Amino group
Imino group
T-A
(Purine-Purine)
results
Depurination (hydrolysis)
Deamination
Mutation hot spot
Repaired
DNA methylation
(regulation of DNA functions,
see epigenetics)
Not repaired
Induced mutation
Some environmental agent = mutagen
– physical - radiation
• Heat
• UV
• Ionizing
– chemicals
• Natural toxins
• Synthetic substances
– Laboratory substances
– Pollutants
– Chemoterapeutics
Natural substances
Psoralen
Aflatoxin
Aspergillus sp.
Laboratory chemicals: acridine orange,
ethidium bromide,
propidium jodide
Fluorescent dyes
BrdU - thymine analogue
A senescent endothelial cell stained with the
fluorescent dye acridine orange to visualise the
lysosomes.
Acrylamide
Polyacrylamide
Pollutants
E.g. benzpyrene
polyaromatic hydrocarbons
(PAH)
Metabolized to epoxides in liver
DNA adduct
Mutation
Biological warfare agent
Mustard gase
Iranian victim (end of 20th century)
I. World war victim (beginning of 20th century)
Correction of DNA errors
• DNA polymerase with
proofreading ability
• Repair mechanisms
nuclear but not
mitochondrial DNA
DNA repair mechanisms
• Direct repair
• Excision repair
the change is reversed
template is needed
no template is needed
mainly in prokaryotes
in eukaryotes
Repair of single strand damage
(complementer strand is used as template)
Xeroderma pigmentosum is caused by the defective
nucleotide excision repair enzymes
Repair of double strand breaks
(DSB)
may result loss of
nucleotides
= deleterious
Sister chromatid (after S phase)
or like in meiosis, homologous
chromosome is used as
template = safety
Multicellular cell cycle
M-phase
cytokinesis
mitosis
M
G2
Checkpoints:
Restriction point
G2
M (spindle)
G1
G2
Restriction
point
S
Interphase
Go
- Growth factors
- anchorange
Function and activity of checkpoint
machinery
G1
G2
DNA damage
not complete DNA replication
M
free kinetochors
sensor
protein kinases
transducer
effector
s t o p
of
c e l l
repair
c y c l e
Ataxia telangiectasia (ATM=sensor)
Its mutation causes rare, neurodegenerative,
inherited disease (AR), that affects many parts
of the body and causes severe disability,
characterized by radiosensitivity
and different tumors.
Role of BRCA (transducer) proteins in
DNA repair
BRCA mutations are found in breast and ovarian tumors.
p53
Site of mutations - in the organism
• Somatic - in somatic cells
localized- inherited within
cells of an organism
(mosaicism: tumors,
antibody diversity, etc.)
higher in dividing cells
• Generative - in primordial
germ line
inherited from one
generation to the next one
And nondisjunction
of sex chromosomes
Site of mutations - in a gene
1
2
UTR
UTR
3
4
1/ Promoter mutations  decreased transcription
2/ Exon mutations  amino acid change or truncated protein (stop) see later
3/ Intron mutations  errors in splicing
4/ Polyadenylation site mutations  decreased mRNA stability
5 5 UTR  disturbed ribosome binding
Mutations of other regulatory sequences (enhancers, silencers) also may influence transcription.
B.R. Korf: Human Genetics and Genomics,2006
Splicing mutations
B.R. Korf: Human Genetics and Genomics,2006
Splicing mutations
B.R. Korf: Human Genetics and Genomics,2006
Different mutations of a gene may lead to
different malfunctions of the protein(=CFTR)
Most frequent
Function and mutations
Back mutation or reversion is a point mutation that restores the original
sequence and hence the original phenotype.
Lethal mutations are mutations that lead to the death of the organisms
which carry the mutations.
Gain-of-function mutations - change the gene product such that it gains
a new and abnormal function. These mutations usually have dominant
phenotypes.
Loss-of-function mutations - gene product having less or no function.
Phenotypes associated with such mutations are most often recessive.
Exception is when the reduced dosage of a normal
gene product is not enough for a normal phenotye
(this is called haploinsufficiency).
Dominant negative mutations - the altered gene
product acts antagonistically to the wild-type allele.
These mutations are characterised by a dominant
phenotype. In humans, dominant negative mutations
have been implicated in cancer (e.g. mutations in
genes p53, ATM).
Fitness and mutations
- Most are neutral – during evolution later may be harmful
or beneficial
- Some are beneficial –
- harmful one mutates back to wild
- getting beneficial function
– diversity of antibody
- CCR532 – HIV resistency
- sickle cell anemia – malaria
resistency
- Some are harmful – causing diseases (all monogenic
inherited diseases)
Size of mutations
•
Large
Genome mutation = change of chromosome number
Cytogenetics
•
Medium
Chromosome mutations = change of chromosome structure
•
Small
gene mutations = ranging from a change of single nucleotide to a whole
gene (not visible)
Affecting the lenght of DNA
Deletion (single base or shorter-longer sequences)
Insertion (single base or shorter-longer sequencesrepetitive
more insertion than deletion
No effect on the length of DNA
nucleotide substitution
Repetitive insertions
– Tandem repeats
• Satellite DNA
– pericentromeric heterochromatin
• Minisatellite (VNTR)
– 10-60 bp
– Telomere
• Microsatellite (STR=short tandem repeats)
– 2- some bp
– good markers of kinship
– Repeat number expansion  diseases
– Interspersed repeats:
• SINEs (Short Interspersed Elements),
• LINEs (Long …) e. g. L1
38
Microsatellite
(STR = short tandem repeats)
• 1-4 bp
• Trinucleotide (triplet) repeats are very frequent
– only few of them cause disease
Trinucleotide repeats may be either in coding(C) or
noncoding (NC) region
NC
C
NC
(huntingtin)
(Huntingtin)
C
C coding
NC noncoding
Polyglutamine
disorders
• CAG repeats
• Neurodegenerative disorders
• Different proteins
• Gain of function mutations
• Variable length
• Expansion
• Replicational slippage
Polyalanine
disorders
Replication slippage
Huntingtin
MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQPPPP
PPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLHRPK
KELSATKKDRVNHCLTICENIVAQSVRNSPEFQKLLGIAHELFLLCSDD...
• 350 kD protein
• ubiquitously expressed
• function unknown
• correlation between repeat
size and age of onset and
the severity of disease
(Huntington chorea)
Huntington
healthy
Polyglutamine
disorders
• CAG repeats
Polyalanine
disorders
• GCX repeats
• Neurodegenerative disorders •Developmental abnormalities
• Different proteins
• Transcription factors
• Gain of function mutations
• Loss of function mutations
• Variable length
• Constant length
• Expansion
• Stable
• Replicational slippage
• Uneven crossing over
Uneven crossing over
Uneven
sister chromatid exchange
Polyalanine disorder
Disorder
• Holoprosencephaly
Gene
ZIC2
Deletion or insertion of a single nucleotide (InDel)
It is a frameshift mutation if number of nucleotide is not a multiple of three,
and in-frame if number of nucleotide is a multiple of three
DNA
mRNA
protein
Mutant protein
Medium InDel mutations
• Deletion
– Pl. Hypodontia
(Deletion of Pax 9)
• Insertion
– (retro)transposons
• Eg. L1 hemophilia A
L1 is a LINE: Long Interspersed Elements
L1 insertion and recombination in Hemophilia A
VIII. Blood cloting
factor gene (F8)
Hemophilia A inversion mutation
due to recombination between L1
repetitive sequences within (gray
arow) an outside (red arrow) the
F8 gene
51
Single nucleotide substitution
Nucleotide substitutions in coding region
Transition
Pyr ↔ Pyr
Pu ↔ Pu
More frequent in human
Transversion
Synonymous
No change in amino acid
Pyr ↔ Pu
not synonymous
change of amino acid or no amino acid
Missense mutation – sickle cell anemia
Frequences of disease causing mutations
Polymorphism
• Polymorphism appears at different levels:
– Phenotype polymorphism
– Protein polymorphism (Immunoglobulins, ABO blood groups)
– Genetic (DNA) polymorphism
Rate of genetic polymorphism
– Identity between individuals = 99,5%
– Difference between individuals = 0,5%
DNA Polymorphism
• It is variation of DNA sequence that is
common in the general population (>1%)
• Most are neutral, but some confer
susceptibility or resistance to disease
• In human genome there are many, that is
why can be used for personal identification
• Detection technics are available
Genetic polymorphism
• chromosomal (minor variants)
• tandem repeats
Satellite DNA
pericentromeric heterochromatin
Minisatellite (VNTR)
Telomere
Microsatellite (STR=short tandem repeats)
• Single nucleotide polymorphism (SNP)
Chromosomal polymorphism
1, 9,16 chromosomes centromeres
Y chromosome long arm
Nucleotide substitution=SNP
(single nucleotide polymorphism)
ATGGTAAGCCTGAGCTGACTTAGCGT
ATGGTAAACCTGAGTTGACTTAGCGT


SNP
SNP
Disease resistant population Disease susceptible population
Genotype all individuals for thousands of SNPs
ATGATTATAG
geneX
ATGTTTATAG
Resistant people all have an ‘A’ at position 4 in geneX,
while susceptible people have a ‘T’
Some disease associated SNPs
Association ≠ correlation
PPARG = Peroxiszóma Proliferátor Aktivátor Receptor γ, APOE = Apolipoprotein E; F5 = Faktor V;
CTLA4 = Cytotoxikus T-lymphocyta Antigén 4; GSTM1 =Glutathione S-transferase Mu 1; INS = inzulin;
KCNJ11 = ATP szenzitív K+csatornát kódoló gén; HF1/CFH = Komplement faktor H;
COL1A1 = Kollagén 1 típus A1; CARD15 = Caspase Recruitment Domain 15;
CNVR = copy number variations
1 kb - 5 megab
About 1500 CNV
12% of genome
2900 gene
is based on mutations and polymorphisms