MUTATION

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MUTATION
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Metabolic Pathways have Topologies
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Different Systems have Different Topologies
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Loss of Function Mutations
Variant 1 2 3 4 5 6
End Product HHEE- -
Build-up CDFGAB
Enzyme E3 E4 E6 E7 E1 E2
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Terms used to describe Genotype-Phenotype
relationships

• Expressivity
• Penetrance
• Pleitropy
• Epistasis
• Allelic Heterogeneity
• Genetic Heterogenity

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Expressivity
The degree of phenotypic expression associated
with a particular allele or genotype.
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Penetrance

• The possibility that someone with a a particular
  genotype will actually express a particular
  phenotype.
• Penetrance P(phenotypegenotype)
• P(genotypephenotype)
• 2. Penetrance can depend on age, body size, sex,
  or other genes and environment.

AA
Often called a variable penetrance
Aa
aa
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Pleiotropy

• The feature of a single variation or polymorphism
  to affect mutlple traits.
• 2. Pleiotropy can occur because the variant
  affects a biochemical pathway with many
  intermediates.
• 3. Pleiotropy can also occur because the effect
  on the biochemical pathways causes higher-level
  disturbances in physiological systems or organ
  systems, which show up as clinical phenotypes as
  well as biochemical phenotypes.

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Epistasis

• Refers to the phenotypic interaction between
  alleles of different genes.
• 2. There are many different types of epistasis.
• 3. Generally, epistasis refers to any type of
  interaction where a genotype at one gene affects
  phenotypic expression of a genotype at a second
  gene.

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Interaction between a regulating gene and its
target
Regulatory Gene
Gene for Active Protein
a-
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A molecular mechanism for recessive epistasis
Dihybrid w/- m/m
Selfed
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3/16 w/- m/m Blocked at second enzyme
w
Enzyme 1
3

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1/16 w/w m/m Blocked at first enzyme
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Genetic Heterogeniety
Variations in mutiple genes are associated with
the same phenotype
Here, G1, G2, G3, and G4 are associated with the
presence or absence of E, the end product.
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• Mutations
• Caused by changes in the DNA sequence
• There are many different categories of mutations
  
• - gametic or somatic
• Phenotypic Effect - recessive, dominant,
  sexlinked, morphological, nutritional,
  behavioral, regulatory, lethal, conditional,
  etc.
• Type of change in the DNA or Protein
• DNA - transition, transversion,
  deletion, insertion
• Protein - silent, neutral, missense,
  nonsense, frameshift

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• Spontaneous Mutation Rates
• Rates are very low (1x10-8 to 1x10-5)
• Rates vary from species to species (lower in
  prokaryotes)
• Rates vary from gene to gene

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• Types of Mutations
• Nucleotide Substitutions
• Insertions Deletions
• Trinucleotide Repeats
• Allelic Expansion and Anticipation

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• Nucleotide Substitutions - involve an alteration
  in the sequence but not the number of nucleotides
  in a gene.
• Missense mutations are single base changes that
  result in the substitution of one amino acid for
  another in the protein product of the gene.
• Nonsense mutations are single base changes that
  create one of the three termination codons in the
  genetic code -- UAA, UAG, or UGA -- resulting in
  a shortened, dysfunctional protein product of
  the gene.

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Insertions and Deletions - involve a change the
number of bases in a gene The impact of an
insertion or deletion in the protein coding
region of the gene can result in a frameshift
mutation.
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Frameshift Mutation
Because codons consist of groups of three bases,
subtracting or adding a base or bases changes the
coding of all the codons that follow.
Consider the following sentence
THE BIG RED FOX If we insert a letter
in the 2nd word, the meaning of everything that
comes after it changes. THE
BIB GRE DFO X
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Cystic Fibrosis Population Frequencies
Source Modified from Cutting GR. Genetic
epidemiology and genotype/phenotype correlations.
In Program and abstracts. NIH Consensus
Development Conference on Genetic Testing for
Cystic Fibrosis. April 14-16, 1997.
http//odp.od.nih.gov/consensus/cons/106/106_state
ment.htm
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• Molecular Basis of Mutations
• Replication mistakes
• A goof once every 10 million bases
• 3'-5' exonuclease allows editing
• Slippage leads to small insertions and deletions
  (frameshifts)
• Caused by Repeated Sequences
• - Fragile-X
• - 50 repeats of CGG is
  normal
• - 50 to 200 is a carrier
  (anticipation)
• - gt200 causes mental
  retardaton
• - Also the cause of
  Huntington's (CAG)
• and Myotonic dystrophy (CTG)

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Error Rates for thermal resistant DNA polymerases
Taq (Thermus aquaticus) 1.1 x 10-4
base substitutions/bp (Tindall and Kunkel,
1988) 2.4 x 10-5 frameshift mutations/bp
(Tindall and Kunkel, 1988) 2.1 x 10-4
errors/bp (Keohavang and Thilly,
1989) 7.2 x 10-5 errors/bp
(Ling et al., 1991) 8.9 x 10-5 errors/bp
(Cariello et al., 1991)
2.0 x 10-5 errors/bp (Lundberg et
al., 1991) 1.1 x 10-4 errors/bp
(Barnes, 1992) http//research.nwfsc.no
aa.gov/protocols/taq-errors.html
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• Mutations due to damage to the DNA template
• 1. Apurinic sites (AP sites) caused by loss of
  the base (the
• glycosidic bond breaks)
• 2. Deamination of cytosine to uracil (or adenine
  to hypoxanthine) usually repaired by
  uracil-DNA-glycosidase
• however, 5-methylcytosine becomes thymidine and
  cant be repaired
• Methylation of cytosine occurs at only specific
  locations
• (only CG in mammals)
• Leads to mutational hot spots
• Many fewer CG's than would be expected

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Mutations due to damage to the DNA template
(continued)

• 3. Radiation
• Ultraviolet light causes thymine dimers.
• Gamma rays and X rays - generate free radicals
  which can not only change bases but can also
  break chromosomes.

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Mutations due to damage to the DNA template
(continued)
4. Chemical mutagens a) base analogs
5-bromouracil can pair
with A or G 2-aminopurine
can pair with T or C b) modifying
agents can cause mispairing unless the damaged
base is removed. - Alkylating
agents (add alkyl groups ) -
Nitrosoguanidine (NG) attacks N7 of
guanine and N3 of adenine -
Ethylmethanesulfonate (EMS) attacks O6 of guanine
- Aflatoxin B1 attacks N7 of guanine - AP
site - Nitrous acid converts NH2
to O (deamination)
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Mutations due to damage to the DNA template
(continued)
4) Chemical mutagens (cont) c) intercalating
agents insert into the DNA between the stacked
bases this causes insertions and deletions
- acridine orange
- proflavin
- ICR-191 5)
Transposons and viruses Genes that jump into
and out of chromosomes that can cause insertions
and deletions.
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DNA Repair

• First line of defense against mutation is to try
  to fix the damaged base.
• a) photoreactivation - in bacteria,
  thymine dimers formed by UV radiation are
  separated by DNA photolyase using visible light
  as the energy source.
• b) O6-methylguanine methyl transferase
  fixes guanines that have been methylated by
  alkylating agents

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DNA Repair
2. Excision repair - remove damaged bases and
use a DNA polymerase to replace them with the
correct bases. a) AP repair - DNA glycosylase
removes damaged bases from the sugar, the
resulting apurinic or apyrimidinic site (AP site)
is removed from the DNA. b) UV damage repair -
excise area around thymine dimer and resynthesize
the strand. 3. Xeroderma pigmentosum - no UV
repair, high skin cancer rates and death by age
30.
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DNA Repair
4. Mismatch repair a)
Enzymes nick DNA on both sides of the mismatch,
DNA polymerase I and DNA ligase do the rest. (How
does one recognize the "right" strand? In E. coli
the "old" DNA is methylated at GATC.) 5.
Recombination repair (post-replication repair)
a) DNA Polymerase III skips the damaged
region, leaving a gap. b) RecA
promotes recombination with the sister duplex
(the damaged region is still there but the DNA is
intact). c) Results in gene conversion.
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Database
Address dbSNP-polymorphism repository
http//www.ncbi.nlm.nih.gov/SNP/ Genome
Database, GDB http//gdbwww.gdb.or
g/ Genatlas
http//www.infobiogen.fr

Gene Cards Database
http//bioinformatics.weizmann.ac.il/cards/ HGBAS
E (Human Genic Bi-Allelic SEquences)
-database of intra-genic DNA sequence
polymorphisms
http//hgbase.interactiva.de Human Gene
Mutation Database,HGMD
http//www.uwcm.ac.uk/uwcm/mg/hgmd0.html