BIOL 588

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BIOL 588 LECTURERS NOTES FOR SEPT 28,
2006 Mutation, genetic disease and
experimental genetics Ken Hastings
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Allelic polymorphism - the existence of diverse
alternative forms of any given gene within a
species. - results from DNA mutation - most
"new"neutral alleles die out, by random drift,
before they can become established in the
population. Rarely, a new allele thrives, by
random drift, and becomes established in the
population. When more than one allele is
established in the population, these alleles are
essentially at war - a war of random drifting,
which will eventually be won by one of them, just
by chance. This is called fixation. The level of
allelic polymorphism in a species is determined
by the dynamic interaction between mutation
(which generates new alleles) and drift/ fixation
(which eliminates pre-existing alleles).
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- some human gene allelic variants are very old,
even predating the species. Others have arisen
within the population since speciation. -
individual diploid organisms contain two copies
(maternal, paternal) of each gene. These two
copies can be identical (homozygous) or two
different alleles (heterozygous).
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Human DNA sequence diversity Overview based on
comparative DNA sequencing data If you randomly
pick from the human population 100 examples of
any given gene, and compare them all at any given
base in the sequence, you will find that at 299
out of 300 bases examined all 100 examples would
have the same base at that position. Sequence
heterogeneity will be observed at 1/300
positions. Thus the average length of DNA
sequence blocks that are identical throughout
the entire human population (where identical
means that any minor alleles that exist are less
than 1 as abundant as the major allele) is
300 bp. The average 10 kb gene would have on the
order of 30 allelic variants whose population
frequency is 1 . The average 1 kb
protein-coding sequence would have 3 such
variants in the population. (based on Cargill
et al (1999) Characterization of
single-nucleotide polymorphisms in coding regions
of human genes. Nature Genetics 22, 231-238)
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When the sample size is only two examples (e.g.
the maternally and paternally inherited alleles
in an individual person) the average length of
sequence blocks that are identical in both
alleles is 2 kb. Thus the two alleles you have of
the average 10 kb gene differ at 5 bases, so you
are heterozygous at most genes. The two alleles
you have of the average 1 kb protein-coding
sequence differ at 0.5 bases. About half of
these DNA differences change the amino acid.
Thus, about 3/4 of your proteins are homogeneous,
but you contain two sequence variants of the
remaining 1/4. (based on Cargill et al (1999)
Characterization of single-nucleotide
polymorphisms in coding regions of human genes.
Nature Genetics 22, 231-238)
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Types of mutation - point mutation single-base
change most common kind of genetic
polymorphism (SNP single nucleotide
polymorphism) - deletion of whole gene or part
of it - insertion - of transposable element
or virus genome - repeat sequence expansion -
duplication of a DNA region or chromosome -
chromosome translocation/inversion (not usually
heritable)
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Allelic polymorphism and phenotypic expression
Allelic polymorphism can be - phenotypically
silent (e.g. mutation in intergenic DNA, or codon
mutation to synonym) - phenotypically neutral
(e.g. human hair or eye color alleles). Gives
rise to inconsequential genetic individuality.
- phenotypically beneficial (very rare). The
new mutant allele provides a selective advantage
to the organism carrying it. Such alleles become
fixed rapidly. This is the basis of adaptive
evolution. - phenotypically deleterious
(common). The new mutant allele confers a
disadvantage on the organism carrying it. This is
genetic disease. These alleles can never be
fixed, but for various reasons (e.g. common
mutation, heterozygote advantage, Hardy-Weinberg
equilibrium) they can be chronic features of the
species' genetic polymorphism. Deleterious
effects could be due to gene dosage (deletion or
duplication), or effects on transcription, RNA
splicing, translation, or protein function or
stability.
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Mechanisms underlying genetic disease inheritance
patterns
dominant - heterozygotes have the disease.
amount of protein
functional threshold
- gene dosage - one-half of the normal amount of
the gene product (from the wild-type allele is
not enough to support normal function..
haploinsufficiency
/
/-
- the mutated protein has gained a new or
uncontrolled function that causes the disease.
(a dangerous "rogue" protein variant)
- the mutant protein is inactive, but interacts
with wild-type protein molecules, e.g. by forming
an inactive oligomer, in such a way as to
interfere with the wild-type protein's function.
This is a type of "dominant negative" mutation.
From Molecular Biology of the Cell, Alberts et
al, 4th ed. Garland Science Fig 8-67
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Mechanisms underlying genetic disease inheritance
patterns (contd)
recessive - only homozygotes have the disease
heterozygotes are normal, but are carriers of the
defective allele.
-the mutant gene/protein is absent/inactive, but
one-half of the normal amount of gene product
(from the other, wild-type allele) is sufficient
for normal function.
- the mutant gene is inactive but protein from
wild-type allele nonetheless accumulates to
near-normal levels (example - a protein A is
unstable unless it forms a complex with a
partner protein B, which is produced limiting
amounts. Normal levels of AB could accumulate
even if the rate of synthesis of A were reduced
to one-half in A/- heterozygotes. )
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Mechanisms underlying genetic disease inheritance
patterns (contd)
X-linked recessive - only males have the disease
and it never appears in their sons, but only in
their daughters' sons. The gene is on the X
chromosome and the mutation is functionally
recessive.
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Disease gene mapping and positional cloning Step
1. Mapping/ cloning Establish genetic linkage
between the disease gene polymorphism and other
previously mapped neutral or silent
polymorphisms. This gives you a genetic map
location with a precision on the order of several
centimorgans which amounts to several million
base-pairs of DNA, which might include 10 or more
genes, one of which is the gene responsible for
the disease. Obtain DNA clones of genes in the
region.
Step 2. Candidate gene assessment. Assess each
gene in the region as a possible candidate. This
is guesswork based on knowledge of the disease
symptoms and on knowledge of the tissue pattern
of expression of each gene in the region, and on
the probable biochemical function of the encoded
proteins, based on sequence homology with broad
functional classes of proteins (e.g. a kinase, an
ATPase, a growth factor receptor, etc etc)
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Disease gene mapping and positional cloning
(contd)
Step 3. Identify pathogenic mutation. Sequence
DNA of plausible candidate genes from affected
and unaffected individuals, looking for a
polymorphism that is associated with affected
individuals, but not unaffected individuals.
(Watch out, there can also be silent and neutral
poymorphisms.)
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Genetic neurological diseases 90 genetic
neurological diseases have been elucidated. Some
in the different general mutation classes are
- point mutation retinitis pigmentosum
(dominant forms) mutated gene encodes rhodopsin
familial amyotrophic lateral sclerosis (some
pedigrees) mutated gene encodes superoxide
dismutase - deletion Duchenne/Becker muscular
dystrophy mutated gene encodes dystrophin -
duplication Charcot Marie Tooth (peroneal
muscular atrophy) duplication of gene encoding
peripheral myelin protein-22 Alzheimer's
associated with Downs syndrome (trisomy 21)
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Trinucleotide repeat mutations In several genes
there exist CAG repeats, i.e. CAGCAGCAGCAG(30-120
bp in length). These can be in protein-coding or
in untranslated sequences. the number of repeats
varies slightly among normal alleles. Alleles
with more than the usual number of repeats are
somewhat unstable and tend to expand, sometimes
catastrophically, in subsequent generations.
Alleles with large repeat numbers are not
functionally normal but are impaired for
gene/protein function. This impairment can be
even greater when huge numbers of repeats (gt1000)
occur. The instability and repeat
length-dependent severity of the functional
impairment account for the phenomenon of "genetic
anticipation", or worsening of the disease from
one generation to the next.
Some trinucleotide repeat neurological
disorders Huntingtons Disease (basal
ganglia) Kennedy Disease (spinal motor
neurons) Myotonic Dystrophy (muscle and
CNS) Fragile X syndrome (mental
retardation) Spinocerebellar ataxia (atrophy of
cerebellum, spinocerebellar tracts
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Three generations in a myotonic dystrophy
family 100, 100, 1000 CAG repeats
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Disease gene cloning medical implications -
carrier detection, prenatal diagnosis - perfect
accuracy theoretically possible within affected
pedigrees - limitation in many diseases many
cases are new mutations. No amount of genetic
screening/ counselling of affected families will
ever eliminate these diseases. One would need to
continuously screen the entire population. -
understanding and controlling pathology -
insight into affected cellular process may
suggest therapeutic strategies (i.e.- treating
the symptoms). - opens door to production of
animal model of disease for preclinical testing
of therapeutic strategies. -cloning of gene
opens door to therapy directed at the cause, not
the symptoms, i.e. gene replacement therapy.
limitations to gene replacement therapy - not
applicable to dominant disorders - in some
cases may require early diagnosis for effective
treatment and hence be unsuitable for diseases
with a high rate of new mutation. - no generally
practical somatic gene introduction technology
has been developed, although some viral vectors
are promising and transplantation of transformed
cells may provide an alternative route.
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Experimental mammalian genetics Altering the
genome by transgene introduction and gene
knockouts. Creating animal models for human
genetic diseases Different strategies for
dominant and recessive disorders. - dominant
disorders (dominant negative or "rogue" protein
types). Introduce gene encoding mutant protein
at a random site in the mouse genome by producing
transgenic mice. Each resulting transgenic mouse
line has two normal mouse alleles and one
transgene locus containing one or, usually more,
serially repeated copies of the mutant human
allele. - recessive disorders. - gene knockout
strategy. Directly inactivate one of the
endogenous normal mouse alleles by targeted
mutation based on homologous recombination. The
first generation of mice are heterozygous and
presumably would be normal (recessive disorder).
Produce homozygous knockout animals by inbreeding
- these may show the disease symptoms. -gene
expression knock-down by antisense or RNAi
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Production of transgenic mice by zygote
pronuclear microinjection
Injection pipet Mouse Holding pipet
inject DNA implant in pseudo pregnant ?
mouse check pups for incorporation of gene by
tail DNA Southern blot breed identified founder
mice to pass on transgene to next generation
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Targeted mutation gene knockouts example from
myogenin knockout - prepare replacement vector
- transfect into ES (embryonic stem) cells -
select for targeted insertion/replacement events
(positive/negative selection) - verify by
Southern blot or PCR - produce chimeras by cell
injection into blastocysts - breed chimera to
look for germ-line chimeras passing mutant gene
on to progeny - breed heterozygotes to produce
homozygous animals
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Gene knock-in mutagenesis A variant of the gene
knock-out approach, also based on targeted
homologous recombination in ES cells. In a
knock-in experiment the targeting vector is
designed so that a targeted insertion event
results in an experimentally useful protein (e.g.
beta-galactosidase) being produced by
transcription/ translation from the promoter of
the targeted gene. The targeting vector construct
would not include a promoter for (e.g.) beta-gal
expression - so beta-gal is not expressed except
when inserted into a gene that provides a
functional promoter. Following targeted insertion
beta-gal (or other experimental protein) is
expressed from the targeted genes promoter. Thus
(e.g.) beta-gal is "knocked in" to the target
gene. This simultaneously mutates the gene (by
substituting the new protein-coding sequence for
the gene's original encoded protein) and provides
an experimentally useful protein expressed in the
same pattern as the targeted gene.
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Conditional transgene expression and conditional
knockouts It may be desirable to artificially
control transgene expression or to limit a gene
knockout to particular tissues or to a particular
phase of the life cycle. For example a dominant
negative mutant transgene may kill the animals
that harbor it during development. If so you will
never be able to produce a transgenic line
carrying that mutant gene. But if you could turn
the gene on and off at will you could make
transgenic lines leaving the gene off, and then,
once you had established the line and made a
population of animals, you could turn the gene on
and study the pathogenic process. Or in the case
of knockout you might wish to target the mutation
to a particular tissue to see if the pathology is
due to the lack of expression in that cell type
and not lack of expression in some other cell
type that interacts biologically.
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Conditional transgene expression Tet on/ off
system permits the experimenter to control the
timing of expression. tetracycline-regulated
transactivator (tTA) is a fusion between the
tet-repressor DNA-binding domain and a VP16
activation domain (from a viral transcription
factor). tTA binds specifically to the tetO
operator and induces transcription from an
adjacent minimal promoter.
You make transgenic mice with a construct in
which the protein-coding sequence of interest is
cloned downstream of a minimal promoter, upstream
of which has been introduced the tetO operator
(in multiple copies). These transgenic mice do
not express the gene because the minimal promoter
is not active.
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To activate gene expression, you breed the
experimental transgene into mice that already
have another transgene that expresses the tTA
protein in some or all tissues. Mice that contain
both the tTA encoding transgene and the
experimental tTA-dependent construct will express
the experimental gene, i.e. the gene is "on" in
that generation of mice. To gain control within
the lifetime of the animal, one can add
tetracycline to the water. Tetracycline binds to
the tTA protein, preventing it from activating
transcription. Thus, you can turn the gene off.
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Cre-lox system for tissue-specific gene knockout.
Cre recombinase from bacteriophage Pl catalyzes
recombination between DNA sites carrying the loxP
sequence (a 34 bp sequence). 1. Make a construct
in which a key exon of the gene you want to knock
out is flanked by loxP sites. Eventually we will
delete that exon by Cre-mediated
recombination/excision. Because the loxP-tagged
gene will be introduced into the germ line by
homologous recombination in ES cells, also
include a neo gene in the construct, also flanked
by loxP sites (use a total of 3 loxP sites). 2.
Select properly targeted ES cells, then express
low levels of Cre by transient transfection of
the ES cells with a Cre-encoding plasmid. This
results in partial Cre recombination, generating
a product in which the neo gene has been excised.
Clone out cells carrying the neo-excised allele,
and make germ-line chimeric mice, and recover
transgenic lines.
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3. Breed the transgenic lines with other lines
carrying trans genes that express Cre recombinase
in the tissue of interest (use tissue-specific
promoter to express Cre protein-coding sequence).
Cre will excise the loxP-flanked exon, knocking
out the gene only in the tissue expressing the
Cre recombinase.
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Application of RNAi to mammalian cells . Make
dsRNA corresponding in sequence to the mRNA you
wish to knock down. Problem Long dsRNAs induce
nonspecific translational arrest in vertebrate
cells. Solutions -introduce ready-made siRNAs
(bypass Dicer). - chemically or enzvmaticallv
synthesized double-stranded 21-mer siRNAs. -
transfect cells with plasmid encoding short
hairpin RNAs (shRNA) recognized and attacked by
Dicer. Continuous production of siRNA via Dicer
in the cell.
Example Use of RNA polIII expression vector
based on promoter of U6 snRNA gene.