Molecular pathology:
Physiopathology effect of
Mutations
Dr Derakhshandeh, PhD
Mutations
• changes to the either DNA or RNA
• caused by copying errors in the genetic
material:
– Cell division
– Ultraviolet
– Ionizing radiation
– chemical mutagens
– Viruses
2
Mutations In multicellular
organisms
• can be subdivided into:
– Germline mutations
• can be passed on to descendants
– Somatic mutations
• cannot be transmitted to descendants in
animals
3
Germ & Somatic cell
• a mutation is present in a germ cell
– can give rise to offspring that carries the
mutation in all of its cells
– Such mutations will be present in all descendants of this
cell
– This is the case in hereditary disease
• a mutation can occur in a somatic cell of an
organism
• certain mutations can cause the cell to become
malignant
– cause cancer
4
Classification
By effect on structure
• Gene mutations have varying effects on
health:
– where they occur
– whether they alter the function of
essential proteins
5
Structurally, mutations can be
classified as:
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Point mutations
• caused by chemicals/malfunction of DNA
replication
• exchange a single nucleotide for another
• Most common is the transition that
exchanges a purine for a purine (A ↔ G)
• or a pyrimidine for a pyrimidine, (C ↔ T)
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Transition
• caused by:
– Nitrous acid
• base mispairing
– 5-bromo-2-deoxyuridine (BrdU):
• mutagenic base analogs
8
Transversion
• Less common
• exchanges a purine for a pyrimidine
• or a pyrimidine for a purine (C/T ↔ A/G)
9
Point mutations that occur within
the protein coding region of a gene
– depending upon what the erroneous codon
codes for:
• Silent mutations:
– which code for the same amino acid
• Missense mutations :
– which code for a different amino acid
• Nonsense mutations :
– which code for a stop and can truncate the
protein
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Insertions
• add one or more extra nucleotides into the DNA
– usually caused by transposable elements
– or errors during replication of repeating
elements (e.g. AT repeats)
• in the non/coding region of a gene may alter:
– splicing of the mRNA (splice site mutation)
– or cause a shift in the reading frame (frame
shift)
• significantly alter the gene product
• Insertions can be reverted by excision of the
Transposable element
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Deletion
• remove one or more nucleotides from
the DNA
• Like insertions, these mutations can
alter the reading frame of the gene
• Delitions of large chromosomal
regions, leading to loss of the genes
within those regions
• They are irreversible
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Deletions/insertions/duplications
• Out of frame
• In frame
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Deletions/insertions/duplications
Out of frame:
result in frameshifts giving rise to stop
codons.
no protein product or truncated protein
product
 deletions/insertions in DMD patients :
truncated dystrophins of decreased
stability
RB1 gene - usually no protein product
in retinoblastoma
14
Deletions/insertions/duplications
In frame:
loss or gain of amino acid(s)
depending on the size and may give
rise to altered protein product with
changed properties
eg CF Delta F508 loss of single
amino acid
In some genes loss or gain of a single
amino acid: mild
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In frame:
In some regions of RB1 a single amino
acid loss:
rise to mild retinoblastoma or
incomplete penetrance
 BMD patients:
Some times in-frame
deletions/duplications
DMD deletions:
 mostly disrupt the reading frame
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Deletions/insertions/duplications
In untranslated regions:
these might affect
transcription/expression and/or stability
of the message:
Fragile X
MD expansions
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Large-scale mutations in
chromosomal structure
18
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Amplifications
(gene duplications)
• leading to multiple copies of all
chromosomal regions
• double-minute chromosomes:
– Sometimes, so many copies of the amplified
region are produced
– they can actually form their own small pseudochromosomes
• increasing the dosage of the genes
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Amplifications
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Chromosomal translocations:
• Fusion genes:
– Mutations: to juxtapose previously
separate pieces of DNA
– potentially bringing together separate
genes to form functionally distinct (e.g.
bcr-abl)
• Chromosomal translocation:
– interchange of genetic parts from
nonhomologous chromosomes
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Interstitial deletions:
• an intra-chromosomal deletion:
– removes a segment of DNA from a single chromosome
– For example, cells isolated from a human
astrocytoma, a type of brain tumor
– have a chromosomal deletion removing sequences
between the "fused in glioblastoma" (fig) gene and
the receptor tyrosine kinase "ros", producing a fusion
protein (FIG-ROS)
– The abnormal FIG-ROS fusion protein has
constitutively active kinase activity
– causes oncogenic transformation (a transformation
from normal cells to cancer cells)
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Astrocytoma & Astrocyte
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Astrocytoma
• a primary tumor of the central nervous system
• develops from the large, star-shaped glial cells known
as astrocytes
• Most frequently astrocytomas occur in the brain
• but occasionally they appear along the spinal cord
• occur most often in middle-aged men
• Symptoms of an astrocytoma, similar to other brain
tumors:
– depend on the precise location of the growth
– For instance, if the frontal lobe is affected
• mood swings and changes in personality may
occur
• a temporal lobe tumor is more typically
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associated with speech and coordination
difficulties
• Chromosomal inversions:
• Reversing the orientation of a
chromosomal segment
• Loss of heterozygosity:
– loss of one allele:
• either by a deletion
• recombination event
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By effect on function
•
•
•
•
Loss-of-function mutations
Gain-of-function mutations
Dominant negative mutations
Lethal mutations
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Loss-of-function mutations
• Wild type alleles typically encode a product
necessary for a specific biological function
• If a mutation occurs in that allele, the
function for which it encodes is also lost
• The degree to which the function is lost can
vary
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Loss-of-function mutations
• gene product having less or no function:
– Phenotypes associated with such mutations are
most often recessive:
– to produce the wild type phenotype!
• Exceptions are when the organism is haploid
• or when the reduced dosage of a normal gene
product is not enough for a normal
phenotype (haploinsufficiency)
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Loss-of-function mutations
• mutant allele will act as a dominant:
• the wild type allele may not
compensate for the loss-of-function
allele
• the phenotype of the heterozygote will
be equal to that of the loss-of-function
mutant (as homozygot)
– to produce the mutant phenotype !
30
Loss-of-function mutations
• Null allele:
– When the allele has a complete loss of function
• it is often called an amorphic mutation
• Leaky mutations:
– If some function may remain, but not at the
level of the wild type allele
• The degree to which the function is lost can vary
31
Gain-of-function mutations
• change the gene product such that it gains a
new and abnormal function
• These mutations usually have dominant
phenotypes
• Often called a neomorphic mutation
• A mutation in which dominance is caused
by changing the specificity or expression
pattern of a gene or gene product, rather
than simply by reducing or eliminating the
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normal activity of that gene or gene product
Gain-of-function mutations
• Although it would be expected that most mutations
would lead to a loss of function
• it is possible that a new and important function could
result from the mutation:
– the mutation creates a new allele:
• associated with a new function
• Any heterozygote containing the new allele
along with the original wild type allele will
express the new allele
• Genetically this will define the mutation as a
dominant
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Dominant negative mutations
• Dominant negative mutations:
– antimorphic mutations
– an altered gene product that acts
antagonistically to the wild-type allele
– These mutations usually result in an
altered molecular function (often
inactive):
• Dominant
• or semi-dominant phenotype
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Dominant negative mutations
• In humans:
– Marfan syndrome is an example of a
dominant negative mutation
– occurring in an autosomal dominant
disease
– the defective glycoprotein product of the
fibrillin gene (FBN1):
» antagonizes the product of the normal
allele
35
Fibrillin gene
36
37
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Lethal mutations
• lead to a phenotype:
– incapable of effective reproduction
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By aspect of phenotype affected
Morphological mutations
• usually affect the outward appearance of an
individual
• Mutations can change the height of a plant
or change it from smooth to rough seeds.
• Biochemical mutations result in lesions
stopping the enzymatic pathway
• Often, morphological mutants are the direct
result of a mutation due to the enzymatic
pathway
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Special classes
Conditional mutation
• wild-type (or less severe) phenotype under certain
"permissive" environmental conditions
• a mutant phenotype under certain "restrictive"
conditions
• For example: a temperature-sensitive mutation
can cause cell death at high temperature
(restrictive condition), but might have no
deletirious consequences at a lower temperature
(permissive condition).
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Nomenclature
• Nomenclature of mutations specify the type of
mutation
• and base or amino acid changes
• Amino acid substitution: (e.g. D111E)
– The first letter is the one letter code of the
wildtype amino acid
– the number is the position of the amino acid
from the N terminus
– the second letter is the one letter code of the
amino acid present in the mutation
– If the second letter is 'X', any amino acid may
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replace the wildtype
Nomenclature
• Amino acid deletion: (e.g. ΔF508)
– The greek symbol Δ or 'delta'
indicates a deletion
– The letter refers to the amino acid
present in the wildtype
– the number is the position from the
N terminus of the amino acid were it
to be present as in the wildtype
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Harmful mutations
• Changes in DNA caused by mutation can cause errors in
protein sequence
– creating partially or completely non-functional proteins
• To function correctly, each cell depends on thousands of
proteins to function in the right places at the right times
• a mutation alters a protein that plays a critical role in the
body
• A condition caused by mutations in one or more genes is
called a genetic disorder
• only a small percentage of mutations cause genetic
disorders
• most have no impact on health
– For example, some mutations alter a gene's DNA base
sequence but don’t change the function of the protein
made by the gene
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DNA repair system
• Often, gene mutations that could cause a genetic
disorder
• repaired by the DNA repair system of the cell
• Each cell has a number of pathways through
which enzymes recognize and repair mistakes in
DNA
• Because DNA can be damaged or mutated in many
ways:
– the process of DNA repair is an important way
in which the body protects itself from disease
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Beneficial mutations
• A very small percentage of all mutations :
– have a positive effect
• lead to new versions of proteins that help an
organism and its future generations better adapt
to changes in their environment:
– For example, a specfic 32 base pair deletion in
human CCR5 (CCR5-32) confers HIV
resistance to homozygotes
– delays AIDS onset in heterozygotes
– The CCR5 mutation is more common in those
of European descent
– One theory for the etiology of the relatively high
frequency of CCR5-32 in the european
population is that it conferred resistance to the
bubonic plaque in mid-14th century Europe 46
Selection at the CCR5 locus
• CCR532/CCR532 homozygotes are
resistant to HIV and AIDS
• The high frequency and wide distribution of
the 32 allele suggest past selection by an
unknown agent
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The Role of the Chemokine Receptor
Gene CCR5 and Its Allele
(del32 CCR5)
• Since the late 1970s
• 8.4 million people worldwide
• including 1.7 million children, have died
of AIDS
• an estimated 22 million people are
infected with human immunodeficiency
virus (HIV)
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CCR5 and Its Allele ( del32 CCR5)
monocyte/macrophage (M),
T-cell line (Tl)
a circulating T-cell (T)
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• Studies of mutagenesis in many
organisms indicate that the majority
(over 90%) of mutations are recessive
to wild type
• If recessiveness represents the 'default'
state, what are the distinguishing
features that make a minority of
mutations give rise to dominant or
semidominant characters?
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molecular and cellular biology to classify
the molecular mechanisms of dominant
mutation
1. reduced gene dosage, expression, or protein
activity (haploinsufficiency)
2. increased gene dosage
3. ectopic or temporally altered mRNA expression
4. increased or constitutive protein activity
5. dominant negative effects
6. altered structural proteins
7. toxic protein alterations
8. new protein functions
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The concepts of dominance &
recessive
• Formulated by Mendel (1965)
• Why are some disease dominant and other
recessive?
• Dominance is not an intrinsic property of a gene
or mutant allele
• Relationship between the phenotypes of 3
genotypes (AA, AB, BB):
– Dominant
– Semi dominant
– Recessive (depending both on its partner allele)
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Semi dominant
• Example of homozygous mutants:
– Thalassemia, Familial
hypercholesterolemia, Achondroplasia
– Phenotype of the homozygote
• More severity than heterozygote
• Huntington:
– True dominant to wild type
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Dominant mutations are much rarer
than recessive ones
• Insertional inactivation by retroviral DNA
in mouse genom:
– 10-20:1 (Rec:Dom)
• Wright et al.:
– Physiology of the gene action
• Fisher et al.:
– Accumulation of modifier alleles at other
loci
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Alga Chlamydomonas
• Usually haploid
• In a diploid background
– Nevertheless : recessive behavior
– Supporting: Wright ‘s theory
• Indeed, diploidy:
– Protects against recessive mutations!
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Why most inborn errors of
metabolism are recessive?
• Metabolic pathway:
– Not critical rate limiting steps
– Not qualitatively altered function
– Perhaps: dominat mutations:
• Developmental malformations
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Recessive to Dominant mutations
• Caenorhabditis elegans (C elegans):
• Recessive mutations at a series of loci
termed smg:
– May alter the behavior of mutations from
recessive to dominant
• It seems: Wt smg: encode proteins :
– Recognize and degrade mutant mRNA
species (surveillance)
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Types of dominant mutation
• Muller (1932) quantitative changes to a preexisting WT character:
• Amorph
• Hypomorph
• Hypermorph
• Antimorph
• neomorph
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Classical genetics & molecular
mechanism
1. reduced gene dosage, expression, or protein
activity (haploinsufficiency)
2. increased gene dosage
3. ectopic or temporally altered mRNA expression
4. increased or constitutive protein activity
5. dominant negative effects
6. altered structural proteins
7. toxic protein alterations
8. new protein functions
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Classical genetics & molecular
mechanism
•
A distinction between (loss of function):
– reduced gene dosage, expression, or
protein activity (haploinsufficiency)
• And (gain of function):
– increased gene dosage
– new protein functions
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Reduced gene dosage, expression, or
protein activity (haploinsufficiency)
• Inactivation of one of a pair of alleles
• It is important groups because of:
– Mutation > loss of function:
• Deletion, Ch Translocation, truncation,…
– Dosage sensitive genes : interesting group:
• Code for tissue specific protein:
– Type I collagene
– globin
– LDL-Receptor
• Regulatory genes:
– PAX3
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Waardenburg Syndrome (PAX3)
•
•
•
•
•
•
•
Deafness
pigmentary anomalies
white forelock
heterochromia iridis
partial albinism,
Prominent broad nasal root
Hypertrichosis of the medial part of the
eyebrows
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heterochromia iridis
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Increased Dosage
• Increase gene dosage to three copies affect
phenotype less than reduction to one copy
(+21, +18, +13, XXY, than X0,…)
• Critical genes are important
• PMP-22: duplication >Charcot-Marie-Tooth
disease:
– Haploinsufficient > different phenotype of
Increased Dosage!
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Increased Dosage in
Charcot-Marie-Tooth disease:
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Ectopic or Temporally altered
mRNA Expression
• Point mutation in g, d, b
• Alters binding of the transacting factor
– Abrogate the normal switch from
expression of :
 g to d and b
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69
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HPFH
as a δβ-globin Disease
• Large deletions at the β-globin locus
• from the region close to the human Aγ gene
to well downstream of the human β-globin
• gene and including deletion of the structural
δ- and β-globin genes
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HPFH
• Heterozygotes:
– a normal level of HbA2
– even higher levels of HbF (15 to 30 %)
• Homozygotes:
– clinically normal
– albeit with reduced MCV and MCH
• Compound heterozygotes with b
thalassemia:
– clinically very mild
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Why mutations of structural
proteins are frequently dominant?
• Admixture of normal and abnormal
structure components will disrupt the
overall structure
• Biochemical analysis:
– Abnormal mRNA
– Cellular processing
– Secretion
– Without mature Fibrills
• Type I Collagen, Fibrillin in Marfan
73
Toxic protein alterations
• Usually missense mutations:
– Cause structural alteration in mono- or
oligomeric proteins
– Disrupt normal function
– Lead to toxic products or precursors
• Sickle cell mutations (hem S, b6Glu>Val)*
• * Although : recessive
• Coinheritance in cis (hem S+ b23Val>Ile)
– Sickling to manifest in the heterozygote!
74
Toxic protein alterations
• Various point mutations in rhodopsin
– Slow degeneration of rod photoreceptor
outer segment
75
New protein functions
• Creation of new , adventageus protein functions
by mutation:
– The life blood the evolution
– Occurs over protracted time scale
– Protein with truly new function: rare
– Usually pathological
– Juxtaposition of domains from different
proteins.
• Generate new function: ABL-BCR (9;22)
Philadelphia translocation
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A gene affecting brain size
Microcephaly (MCPH)
• Small (~430 cc v
~1,400 cc) but
otherwise ~normal
brain, only mild
mental retardation
• MCPH5 shows
Mendelian autosomal
recessive inheritance
• Due to loss of activity
of the ASPM gene
ASPM-/ASPM-
control
Bond et al. (2002) Nature Genet. 32, 316-320
77
Other mechanism
• Genomic imprinting:
• If a gene is transcribed only from the ch
originating from one of the two parents
• The locus is hemizygous
• Mutation of the allele on the active
chromosome
– Inactive the locus
• Mutation of the other chromosome
– No phenotypic effect
• Beckwith-wiedermann syndrome
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Beckwith-wiedermann
syndrome (BWS)
• The incidence of BWS :
– 1:13700 live births
• The increased risk of tumor formation in
BWS patients:
– 7.5%
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