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MUTATION AND POLYMORFISM

Febr.19. 2016
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
(having no effect on phenotype)
formerly:
• mutation (or mutant) is the variant (allele)
(formerly: if its frequency is ‹ 1 %
in the population / disease causing, it has a
negative connotation)

Human Genom Variation Society offered:


„sequence variant„ or vagy „allelic variant”
Mutation: somatic mutations or germline mutations (recent)
Polymorphism: in a population when the frequency is known
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 Not repaired

DNA methylation
(regulation of DNA functions,
see epigenetics)
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, Fluorescent dyes
ethidium bromide,
propidium jodide
BrdU - thymine analogue

A senescent endothelial cell stained with the


fluorescent dye acridine orange to visualise the
lysosomes.

Acrylamide Polyacrylamide
Pollutants

E.g. benzpyrene Metabolized to epoxides in liver


polyaromatic hydrocarbons
(PAH)

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 are


limited tothe nuclear
but not mitochondrial
DNA
DNA repair mechanisms

• Direct repair • Excision repair


the change is reversed template is needed
no template is needed in eukaryotes
mainly in prokaryotes
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
mitosis cytokinesis

M
G1
G2 Go
G2
Restriction
point
- Growth factors
- anchorange

Checkpoints: S
Restriction point
G2 Interphase
M (spindle)
Function and activity of
checkpoint machinery
G1 G2 M

DNA damage free kinetochors


not complete DNA replication

sensor
protein kinases

transducer

effector

s t o p of c e l l c y c l e

repair
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 may be
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 sequences-


repetitive
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

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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

NC

(huntingtin)
(Huntingtin)
C
C coding
NC noncoding
Polyglutamine Polyalanine
disorders disorders
• CAG repeats

• Neurodegenerative disorders

• Different proteins

• Gain of function mutations

• Variable length

• Expansion

• Replicational slippage
Replication slippage
Huntingtin

MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQPPPP

PPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLHRPK

KELSATKKDRVNHCLTICENIVAQSVRNSPEFQKLLGIAHELFLLCSDD...

• 350 kD protein
• ubiquitously expressed
• function unknown Huntington

• correlation between
repeat size and age of
onset and the severity of
disease (Huntington healthy

chorea)
Polyglutamine Polyalanine
disorders disorders

• CAG repeats • 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 Gene

• Holoprosencephaly 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 arrow) an outside (red
arrow) the F8 gene

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Single nucleotide substitution
Nucleotide substitutions in coding region

Pyr ↔ Pyr
Transition Pu ↔ Pu

More frequent in human

Transversion Pyr ↔ Pu

Synonymous not synonymous


No change in amino acid 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
 Itis variation of DNA sequence that is
common in given population

 Mostare 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 = Peroxisome Proliferator Activator Receptor γ, APOE = Apolipoprotein E; F5 = Factor V;


CTLA4 = Cytotoxic T-lymphocyte Antigene 4; GSTM1 =Glutathione S-transferase Mu 1; INS = insulin;
KCNJ11 = ATP sensitive K+channel coding gene; HF1/CFH = Complement factor H;
COL1A1 = Collagen type A1; CARD15 = Caspase Recruitment Domain 15;
CNV = copy number variations

1 kb - 5 megab
About 1500 CNV
12% of genome
2900 gene
is based on mutations and polymorphisms
Giude to point mutations

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