15
Gene Mutation and
Molecular Medicine
15 Gene Mutation and Molecular Medicine
15.1 What Are Mutations?
15.2 What Kinds of Mutations Lead to
Genetic Diseases?
15.3 How are Mutations Detected and
Analyzed?
15.4 How Is Genetic Screening Used to
Detect Diseases?
15.5 How Are Genetic Diseases
Treated?
15 Gene Mutation and Molecular Medicine
A mutation in a bone marrow cell causes
white blood cells to divide continuously,
resulting in a type of leukemia.
The protein encoded by the mutated gene
stimulates cell division. A chemical has
been found to bind and inactivate this
protein.
Opening Question:
Are there other targeted therapies directed to
specific types of cancer?
15.1 What Are Mutations?
A mutation is a change in the
nucleotide sequence of DNA that can
be passed on to the next generation.
Some mutations arise when DNA
polymerase makes errors that are not
corrected.
15.1 What Are Mutations?
Two types of mutations:
• Somatic mutations: occur in somatic
(body) cells. Passed to daughter cells
in mitosis but not to sexually produced
offspring
• Germ line mutations: occur in germ
line cells that give rise to gametes. A
gamete passes these mutations on at
fertilization
15.1 What Are Mutations?
Mutations affect phenotypes:
• Silent mutation: usually don’t affect
protein function
 May be in a non-coding region, or
code for the same amino acid as
the original
 Common; a result in genetic
diversity that isn’t expressed
15.1 What Are Mutations?
• Loss of function mutations: gene
may not be expressed at all, or protein
doesn’t function
 It is nearly always recessive
15.1 What Are Mutations?
• Gain of function mutation: leads to
a protein with altered function
 Usually dominant
 Common in cancer—the new
protein may stimulate cell division
Figure 15.1 Mutation and Phenotype
15.1 What Are Mutations?
• Conditional mutation: phenotype is
altered only under certain (restrictive)
conditions, (e.g., a protein may be
unstable at high temperatures)
 Mutation is not detectable under
permissive conditions
 Example—point restriction
phenotype in Siamese cats
15.1 What Are Mutations?
• Reversion mutation: most mutations
can be reversed by mutating a second
time
 DNA reverts to the original
sequence
 The phenotype goes back to wild
type
15.1 What Are Mutations?
• Point mutation: change in a single
nucleotide
 This results from the gain, loss, or
substitution of a single nucleotide
 There are two types of base
substitutions—transition and
transversion
15.1 What Are Mutations?
Transition: substitution of one purine
for the other, or one pyrimidine for the
other
15.1 What Are Mutations?
Transversion: substitution of a purine
for a pyrimidine, or vice versa
15.1 What Are Mutations?
A point mutation in the coding region of
a gene will alter the mRNA sequence,
and may or may not result in a change
in the protein.
Silent mutations do not alter amino acid
sequences.
Figure 15.2 Point Mutations (Part 1)
Figure 15.2 Point Mutations (Part 2)
15.1 What Are Mutations?
• Missense mutations: result in
substitution of one amino acid for
another in a protein
 Example: sickle-cell disease.
Sickle allele differs from normal
allele by one base pair, which
alters one subunit of hemoglobin
 Homozygous recessives have
defective, sickle-shaped red blood
cells
Figure 15.2 Point Mutations (Part 1)
Figure 15.2 Point Mutations (Part 3)
Figure 15.3 Sickle and Normal Red Blood Cells
15.1 What Are Mutations?
Missense mutations may have no effect
on protein function.
Or, the protein functional efficiency may
be reduced, but not completely
inactivated.
15.1 What Are Mutations?
Gain of function missense mutations
can also occur:
TP53 codes for a tumor suppressor,
but certain mutations cause the
protein to promote cell division and
prevent cell death.
The TP53 protein gains an oncogenic
(cancer-causing) function.
15.1 What Are Mutations?
• Nonsense mutations: a base
substitution causes a stop codon to
form somewhere in the mRNA
 This results in a shortened protein,
which is usually not functional
 If it’s near the 3' end, it may have
no effect
Figure 15.2 Point Mutations (Part 1)
Figure 15.2 Point Mutations (Part 4)
15.1 What Are Mutations?
• Frame-shift mutations: insertions or
deletions of bases
 These mutations alter the readingframe for the 3-base codons during
translation
 Nonfunctional proteins are
produced
Figure 15.2 Point Mutations (Part 1)
Figure 15.2 Point Mutations (Part 5)
15.1 What Are Mutations?
Chromosomal mutations result in
extensive changes in DNA.
DNA molecules can break and rejoin.
This can be caused by damage to
chromosomes by mutagens or by
errors in chromosome replication.
15.1 What Are Mutations?
Chromosomal mutations:
• Deletions—chromosome may break
in two places and rejoin, leaving out
part of the DNA
• Duplications—can occur with
deletions when homologous
chromosomes break at different
places
Figure 15.4 Chromosomal Mutations
15.1 What Are Mutations?
Chromosomal mutations:
• Inversions—chromosome breaks and
rejoins, with one segment flipped
• Translocations—segment of DNA
breaks off and attaches to another
chromosome; can cause duplications
and deletions
Down syndrome is caused by
translocation of chromosome 21.
15.1 What Are Mutations?
Translocations can involve reciprocal
exchanges of chromosome segments,
as in chronic myelogenous leukemia
(CML).
15.1 What Are Mutations?
Retroviruses insert their DNA into the
host genome at random.
If the insertion is within a gene, it can
cause a loss of function mutation.
The viral DNA can remain in the host
genome and be passed from one
generation to the next. It’s called an
endogenous retrovirus.
15.1 What Are Mutations?
Transposons (transposable elements)
also insert themselves into genes and
cause mutations.
They can move from one position in a
genome to another, and usually carry
genes to encode enzymes for this
movement.
Short sequences can be left behind
and become mutations.
15.1 What Are Mutations?
Some transposons replicate and the
copies are inserted into new sites in
the genome.
Some genomic DNA is sometimes
carried along with the transposon
when it moves, resulting in gene
duplication.
These gene duplication events play an
important role in evolution.
15.1 What Are Mutations?
Mutations are caused in two ways:
• Spontaneous mutations occur with
no outside influence, and are
permanent (movement of
transposons, imperfect cellular
processes)
• Induced mutations are due to
outside agents, or mutagens such as
chemicals or radiation, or retroviruses
Figure 15.5 Spontaneous and Induced Mutations (Part 1)
15.1 What Are Mutations?
Spontaneous mutation mechanisms:
• The four bases can exist in different
forms (tautomers). One form is rare
If a base forms its rare tautomer, it
can pair with the wrong base,
resulting in a point mutation.
15.1 What Are Mutations?
• Chemical reactions may change
bases
Example: loss of an amino group
(deamination) from cytosine.
If not repaired, DNA polymerase will
add an A instead of G.
15.1 What Are Mutations?
• Errors in replication by DNA
polymerase
Most errors are repaired by the
proofreading function, but some
become permanent.
• Imperfect meiosis: nondisjunction and
random breaking and rejoining of
chromosomes
15.1 What Are Mutations?
Induced mutation mechanisms:
• Chemicals can alter bases
Example: nitrous acid can deaminate
cytosine and convert it to uracil, which
has the same result as spontaneous
deamination.
15.1 What Are Mutations?
• Some chemicals add other groups to
bases
Example: benzopyrene in cigarette
smoke adds a chemical group to
guanine and prevents base pairing.
DNA polymerase will add any base at
that point.
15.1 What Are Mutations?
• Radiation damages DNA
Ionizing radiation (X-rays, gamma
rays, radiation from unstable isotopes)
creates highly reactive free radicals.
Free radicals can change bases into
forms not recognized by DNA
polymerase.
15.1 What Are Mutations?
Ionizing radiation can also break the
sugar-phosphate bonds of DNA,
causing chromosomal abnormalities.
UV radiation (from sun or tanning beds)
is absorbed by thymine, causing it to
form covalent bonds with adjacent
bases and disrupt DNA replication.
Figure 15.5 Spontaneous and Induced Mutations (Part 2)
15.1 What Are Mutations?
Mutagens may be human-made or natural.
Plants make many small molecules for
various functions, including defense; some
are mutagenic and carcinogenic.
Nitrites are human-made mutagens (used to
preserve meats). They are converted to
nitrosamines in the smooth ER and can
deaminate cytosine.
15.1 What Are Mutations?
Aflatoxin is made by the mold
Aspergillus.
When mammals ingest the mold, it is
converted by the ER into a product
that, like benzopyrene, binds to
guanine.
15.1 What Are Mutations?
Radiation can be human-made or
natural.
Isotopes from nuclear reactors and
bombs can increase mutation rates.
Natural UV radiation in sunlight can
cause mutations.
15.1 What Are Mutations?
In normal circumstances, DNA damage
occurs daily—about 16,000 events per
cell per day in humans.
About 80% of these are repaired.
15.1 What Are Mutations?
Some base pairs are more vulnerable
than others to mutation.
Cytosine is often methylated at the 5ʹ
position. If 5ʹ-methylcytosine loses an
amino acid, it becomes thymine.
During mismatch repair it is repaired
correctly only half of the time.
Figure 15.6 5´-Methylcytosine in DNA Is a “Hot Spot” for Mutations
15.1 What Are Mutations?
Mutations can have benefits:
Provide genetic diversity for natural
selection.
• Mutations in somatic cells may benefit
an organism immediately
• Mutations in germ line cells may
cause an advantageous change in the
offspring’s phenotype
15.1 What Are Mutations?
Gene duplication arises through
transposon movements or
chromosome rearrangement.
It is not always harmful; and is a source
of genetic variation.
One gene may continue in its original
role while the other may acquire a
gain of function mutation.
15.1 What Are Mutations?
In genes whose products are needed
for normal cell processes, mutations
are often deleterious, especially in
germ line cells.
Offspring can inherit harmful recessive
alleles in the homozygous condition.
Such mutations can produce lethal
phenotypes.
15.1 What Are Mutations?
Somatic cell mutations can also be
harmful.
Mutations in oncogenes can result in
uncontrolled cell division; loss of
function mutations in tumor
suppressor genes prevent the
inhibition of cell division.
15.1 What Are Mutations?
A major public health policy goal is to
reduce the effects of mutagens on
human health.
• The Montreal Protocol bans ozonedepleting chemicals
• Bans on cigarette smoking have
spread throughout the world
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Mutations are often expressed
phenotypically as proteins that differ
from normal (wild-type) proteins.
Abnormalities in enzymes, receptor
proteins, transport proteins, structural
proteins, and others have all been
implicated in genetic diseases.
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Loss of enzyme function:
Phenylketonuria (PKU) results from an
abnormal enzyme, phenylalanine
hydroxylase (PAH).
It normally catalyzes conversion of
dietary phenylalanine to tyrosine.
Loss of the enzyme function causes
phenylalanine and phenylpyruvic acid
to accumulate.
Figure 15.7 One Gene, One Enzyme
15.2 What Kinds of Mutations Lead to Genetic Diseases?
In the PAH gene researchers have
found more than 400 different
mutations that cause PKU.
The mutant alleles are recessive; one
functional allele can produce enough
functional PAH to prevent the disease.
Table 15.1
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Abnormal hemoglobin:
Hemoglobin has four globin subunits,
two α chains and two β chains.
In sickle-cell disease, one amino acid in
the β-globin polypeptide is abnormal.
The abnormal protein forms needlelike
aggregates in the red blood cells,
resulting in sickle-shaped cells.
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Ability of the blood to carry oxygen is
impaired, and the sickled cells block
capillaries, leading to tissue damage.
Hemoglobin has been well-studied, and
hundreds of amino acid substitutions
have been documented.
Many do not alter the function of
hemoglobin.
Figure 15.8 Hemoglobin Polymorphism
Table 15.2 Some Human Genetic Diseases
Examples of inherited diseases caused
by specific protein defects:
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Point mutations:
In sickle-cell disease, all people with
the disease have the same genetic
mutation.
In other diseases, such as PKU, many
different loss of function mutations in
a gene can lead to the disease.
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Large deletions:
Deletions in the X chromosome that
include the gene for muscle protein
dystrophin result in Duchenne
muscular dystrophy.
In some cases, only part of the gene is
missing, leading to a partly functional
protein. Or deletions may include
other genes as well.
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Chromosomal abnormalities:
Gain or loss of complete chromosomes
(aneuploidy) or segments.
Fragile-X syndrome is a restriction in
the tip of the X chromosome that can
result in mental retardation.
Figure 15.9 A Fragile-X Chromosome at Metaphase
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Expanding triplet repeats
The gene responsible for fragile-X
syndrome (FMR1) has a repeated
triplet, CGG, in the promoter region.
This triplet is repeated 6 to 54 times in
normal people, but 200 to 2000 times
in mentally retarded people with
fragile-X.
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Males carrying a moderate number of
repeats (55–199) show no symptoms
and are called premutated.
These repeats increase as daughters
of these men pass the chromosome to
their children.
Increased methylation of cytosines in
the triplets inhibits transcription of
FMR1.
Figure 15.10 The CGG Repeats in the FMR1 Gene Expand with Each Generation
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Normally, FMR1 protein binds to
mRNAs involved in neuron function
and regulates their translation.
Without FMR1 these mRNAs are not
properly translated, and nerve cells
die, resulting in mental retardation.
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Expanding triplet repeats are involved
in other diseases, such as myotonic
dystrophy and Huntington’s disease.
How the repeats expand is not known.
One hypothesis: DNA polymerase
slips after copying a repeat and then
falls back to copy it again.
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Mutations in somatic cells can lead to
cancer. More than two mutations are
usually needed.
The gene mutations leading to each
stage of colon cancer have been
identified.
Three tumor suppressor genes and one
oncogene must be mutated in
sequence in a cell in the colon lining.
Figure 15.11 Multiple Somatic Mutations Transform a Normal Colon Epithelial Cell into a Cancer
Cell (Part 1)
Figure 15.11 Multiple Somatic Mutations Transform a Normal Colon Epithelial Cell into a Cancer
Cell (Part 2)
15.2 What Kinds of Mutations Lead to Genetic Diseases?
Many phenotypes, including diseases,
are multifactorial—caused by
interactions of many genes and
proteins and the environment.
Susceptibility to disease is determined
by these complex interactions.
60% of people are affected by diseases
that are genetically influenced.
15.3 How Are Mutations Detected and Analyzed?
Molecular genetics determines specific
DNA changes that lead to specific
protein changes.
DNA sequencing technology has
allowed entire genomes to be
sequenced. Comparisons with closely
related species allows identification of
mutations.
15.3 How Are Mutations Detected and Analyzed?
Bacteriophages (viruses) attack
bacteria and inject their DNA into the
host cell, causing the cell to produce
more virus particles.
Bacterial defenses include restriction
enzymes that cut DNA into smaller,
noninfectious fragments.
15.3 How Are Mutations Detected and Analyzed?
Restriction enzymes break DNA
backbone bonds between the 3′
hydroxyl group of one nucleotide and
the 5′ phosphate group of the next
(restriction digestion).
Each type of restriction enzyme cuts
DNA at specific sequences—the
restriction site or recognition
sequence.
Figure 15.12 Bacteria Fight Invading Viruses by Making Restriction Enzymes
15.3 How Are Mutations Detected and Analyzed?
Bacterial restriction enzymes can be
isolated and used in the laboratory to
identify DNA sequences of other
organisms.
The enzyme EcoRI cuts DNA at the
following paired sequence:
5ʹ. . . GAATTC . . . 3ʹ
3ʹ. . . CTTAAG . . . 5ʹ
15.3 How Are Mutations Detected and Analyzed?
EcoRI cuts the two strands
simultaneously between the G and the
A of each strand:
15.3 How Are Mutations Detected and Analyzed?
Restriction enzyme digestion is used to
identify mutations.
The DNA fragments must be separated
to identify where the cuts were made.
Restriction sites are not at regular
intervals, so the fragments are
different sizes and can be separated
by gel electrophoresis.
15.3 How Are Mutations Detected and Analyzed?
A mixture of fragments is placed in a
well in a semisolid gel.
An electric field is applied to the gel.
The DNA fragments are negatively
charged, and move towards the
positive end.
Smaller fragments move faster than
larger ones, forming bands.
Figure 15.13 Separating Fragments of DNA by Gel Electrophoresis
15.3 How Are Mutations Detected and Analyzed?
Gel electrophoresis gives 3 types of
information:
• The number of fragments
• The sizes of the fragments
• The relative abundance of the
fragments, indicated by the intensity
of the band
15.3 How Are Mutations Detected and Analyzed?
DNA fingerprinting uses restriction
digestion and gel electrophoresis to
identify individuals based on
differences in their DNA sequences.
It works best with highly polymorphic
sequences—having multiple alleles
that are likely to differ between
individuals.
15.3 How Are Mutations Detected and Analyzed?
Two types of polymorphisms are used:
• Single nucleotide polymorphisms
(SNPs)—inherited variations in a
single base (point mutations).
If a SNP occurs in a restriction
enzyme recognition site, and one
variant isn’t recognized by the
enzyme, then individuals can be
distinguished.
15.3 How Are Mutations Detected and Analyzed?
• Short tandem repeats (STRs)—short
repetitive sequences, usually in
noncoding regions, that are inherited.
PCR is used to amplify fragments
containing STRs. The fragments are
different lengths and can be
separated by gel electrophoresis.
Figure 15.14 DNA Fingerprinting with Short Tandem Repeats
15.3 How Are Mutations Detected and Analyzed?
The FBI uses 13 STR loci in its
combined DNA Index System
(CODIS) database.
With all the alleles and 13 loci, the
probability of two people sharing the
same alleles is very small.
DNA samples from a crime scene can
determine whether a particular
suspect left that sample at the scene.
Table 15.3
15.3 How Are Mutations Detected and Analyzed?
Previously, disease-causing mutations
were discovered by first identifying the
protein involved, then determining the
gene mutation (PKU and sickle-cell).
Reverse genetics: the gene is
identified first, then the protein is
isolated.
Cystic fibrosis: mutant form of CFTR
was isolated, then the protein was
identified.
15.3 How Are Mutations Detected and Analyzed?
Genetic markers are reference points
for gene isolation. Linkage analysis
allows the genes to be identified.
STRs and SNPs are types of genetic
markers.
To narrow down the location of a gene,
a genetic marker that is always
inherited with the gene must be found.
Figure 15.15 DNA Linkage Analysis
15.3 How Are Mutations Detected and Analyzed?
Genes that are always inherited
together in a family must be closely
linked.
Once a linked DNA region is identified,
many methods are available to
identify the actual gene responsible
for a disease.
15.3 How Are Mutations Detected and Analyzed?
DNA technology has potential to help
identify species and varieties.
The DNA barcode project hopes to
identify all species based on one gene
sequence, part of the gene for
cytochrome oxidase.
15.3 How Are Mutations Detected and Analyzed?
The gene for cytochrome oxidase
mutates often and there are many
variations.
A sequence of 650 to 750 base pairs in
this gene is being sequenced for all
organisms.
Figure 15.16 A DNA Barcode
15.4 How Is Genetic Screening Used to Detect Diseases?
Genetic screening: tests to determine
if a person has a genetic disease, is
predisposed, or is a carrier—
• Prenatal screening
• Screening of newborns
• Screening asymptomatic people who
have relatives with genetic diseases
15.4 How Is Genetic Screening Used to Detect Diseases?
Screening may involve analysis for
abnormal protein function.
Newborns are screened for PKU and
treatment can be started immediately.
A simple, rapid blood test for PKU was
developed in 1963.
Newborns are now screened for up to
35 genetic diseases.
Figure 15.17 Genetic Screening of Newborns for Phenylketonuria
15.4 How Is Genetic Screening Used to Detect Diseases?
DNA testing is direct analysis of DNA
for mutations—the most accurate way
of detecting abnormal alleles.
Any cell in the body may be analyzed,
and PCR amplification means that
only a few cells are needed.
15.4 How Is Genetic Screening Used to Detect Diseases?
Fetal cells may be screened before
implantation for diseases such as
cystic fibrosis.
After implantation, fetal cells can be
analyzed by chorionic villus sampling
or amniocentesis.
New methods allow DNA testing of fetal
cells that released into the mother’s
blood.
15.4 How Is Genetic Screening Used to Detect Diseases?
DNA hybridization can be used to
detect a specific sequence such as a
mutation.
PCR is used to amplify a region where
the sequence might occur.
A short synthetic DNA strand called an
oligonucleotide probe is then
hybridized with the denatured PCR
products. The probe is labeled with
radioactivity or a fluorescent dye.
Figure 15.18 DNA Testing by Allele-Specific Oligonucleotide Hybridization
15.5 How Are Genetic Diseases Treated?
Two main approaches to treating
genetic diseases:
• Modify the disease phenotype
• Replace the defective gene
15.5 How Are Genetic Diseases Treated?
Modifying the disease phenotype can
be done in three ways:
1. Restrict the substrate of a deficient
enzyme—as in PKU, reducing
phenylalanine in the diet
2. Metabolic inhibitors, e.g. the inhibitor
used to treat Kareem Abdul-Jabar’s
chronic myelogenous leukemia
(molecular medicine)
15.5 How Are Genetic Diseases Treated?
3. Supply the missing protein
In hemophilia A, blood factor VIII is
missing and blood clotting is impaired.
Clotting proteins are now produced by
recombinant DNA technology.
Figure 15.19 Strategies for Treating Genetic Diseases
15.5 How Are Genetic Diseases Treated?
In gene therapy, the aim is to supply the
missing allele(s) by inserting a new gene
that will be expressed in the host.
• Germ line gene therapy: new gene is
inserted into a gamete or fertilized egg
All adult cells will carry the new gene.
Ethical considerations preclude its use in
humans.
15.5 How Are Genetic Diseases Treated?
• In vivo gene therapy: gene is inserted
directly into a patient cells
Example: lung cancer treatment in
which a solution with a therapeutic
gene is squirted onto a tumor.
15.5 How Are Genetic Diseases Treated?
One challenge: getting the new gene
into the cell.
Genes may be inserted into a carrier
virus, such as adeno-associated
virus.
This has been used to treat Parkinson’s
disease, which is cause by deficiency
of a neurotransmitter.
Figure 15.20 Gene Therapy
15 Answer to Opening Question
Tamoxifen is used to treat some types
of breast cancer—it binds to estrogen
receptors on cancer cells that are
abnormally sensitive to estrogen.
Erlotinib binds to receptors for
epidermal growth factor that are
expressed on some types of cancer
cells.
Working with Data 15.1: Gene Therapy for Parkinson’s Disease
In the test of gene therapy for
Parkinson’s disease, one group of
patients was injected with a solution
containing the adeno-associated virus
with an inserted gene.
The gene codes for an enzyme that
produces the neurotransmitter GABA.
Working with Data 15.1: Gene Therapy for Parkinson’s Disease
A second group of patients received an
injection with no virus.
Over a period of months, the patients
were assessed for changes in motor
function.
Reduction in the rating scale indicates
improvement of function.
Working with Data 15.1, Figure A
Working with Data 15.1: Gene Therapy for Parkinson’s Disease
Question 1:
Compare the control (sham) group
with the gene therapy group.
Were there any differences in their
UPDRS scores?
If so, when were the differences
initially apparent?
Were the differences statistically
significant? How can you tell?
Working with Data 15.1: Gene Therapy for Parkinson’s Disease
Question 2:
Why do you think the score for the
control group changed after the sham
treatment?
Working with Data 15.1: Gene Therapy for Parkinson’s Disease
A second way to assess symptoms is
the global rating of Parkinsonism, in
which the patients are asked to
evaluate their own symptoms.
In this case, an increase in the score
indicates improvement. The results
are shown in Figure B.
Working with Data 15.1, Figure B
Working with Data 15.1: Gene Therapy for Parkinson’s Disease
Question 3:
How did the two groups of patients
compare in their own evaluation of
their symptoms?
How does the fact that this was a
double-blind study influence the
strength of your conclusions?