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Molecular pathology: Physiopathology effect of Mutations

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Title: Molecular pathology: Physiopathology effect of Mutations


1
Molecular pathologyPhysiopathology effect of
Mutations
  • Dr Derakhshandeh, PhD

2
Mutations
  • changes to the either DNA or RNA
  • caused by copying errors in the genetic material
  • Cell division
  • Ultraviolet
  • Ionizing radiation
  • chemical mutagens
  • Viruses

3
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

4
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

5
ClassificationBy effect on structure
  • Gene mutations have varying effects on health
  • where they occur
  • whether they alter the function of essential
    proteins

6
Structurally, mutations can be classified as
7
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)

8
Transition
  • caused by
  • Nitrous acid
  • base mispairing
  • 5-bromo-2-deoxyuridine (BrdU)
  • mutagenic base analogs

9
Transversion
  • Less common
  • exchanges a purine for a pyrimidine
  • or a pyrimidine for a purine (C/T ? A/G)

10
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

11
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

12
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

13
Deletions/insertions/duplications
  • Out of frame
  • In frame

14
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

15
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

16
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

17
Deletions/insertions/duplications
  • In untranslated regions
  • these might affect transcription/expression
    and/or stability of the message
  • Fragile X
  • MD expansions

18
  • Large-scale mutations in chromosomal structure

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20
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
    pseudo-chromosomes
  • increasing the dosage of the genes

21
Amplifications
22
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

23
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)

24
Astrocytoma Astrocyte
25
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
    associated with speech and coordination
    difficulties

26
  • Chromosomal inversions
  • Reversing the orientation of a chromosomal
    segment
  • Loss of heterozygosity
  • loss of one allele
  • either by a deletion
  • recombination event

27
By effect on function
  • Loss-of-function mutations
  • Gain-of-function mutations
  • Dominant negative mutations
  • Lethal mutations

28
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

29
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)

30
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 !

31
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

32
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 normal activity of
    that gene or gene product

33
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

34
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

35
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

36
Fibrillin gene
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39
Lethal mutations
  • lead to a phenotype
  • incapable of effective reproduction

40
By aspect of phenotype affectedMorphological
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

41
Special classesConditional 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).

42
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
    replace the wildtype

43
Nomenclature
  • Amino acid deletion (e.g. ?F508)
  • The greek symbol ? 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

44
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 dont change the function of
    the protein made by the gene

45
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

46
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

47
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

48
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)

49
CCR5 and Its Allele ( del32 CCR5)
monocyte/macrophage (M),
T-cell line (Tl)
a circulating T-cell (T)
50
  • 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?

51
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

52
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)

53
Semi dominant
  • Example of homozygous mutants
  • Thalassemia, Familial hypercholesterolemia,
    Achondroplasia
  • Phenotype of the homozygote
  • More severity than heterozygote
  • Huntington
  • True dominant to wild type

54
Dominant mutations are much rarer than recessive
ones
  • Insertional inactivation by retroviral DNA in
    mouse genom
  • 10-201 (RecDom)
  • Wright et al.
  • Physiology of the gene action
  • Fisher et al.
  • Accumulation of modifier alleles at other loci

55
Alga Chlamydomonas
  • Usually haploid
  • In a diploid background
  • Nevertheless recessive behavior
  • Supporting Wright s theory
  • Indeed, diploidy
  • Protects against recessive mutations!

56
Why most inborn errors of metabolism are
recessive?
  • Metabolic pathway
  • Not critical rate limiting steps
  • Not qualitatively altered function
  • Perhaps dominat mutations
  • Developmental malformations

57
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)

58
Types of dominant mutation
  • Muller (1932) quantitative changes to a
    pre-existing WT character
  • Amorph
  • Hypomorph
  • Hypermorph
  • Antimorph
  • neomorph

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Classical genetics molecular mechanism
  • reduced gene dosage, expression, or protein
    activity (haploinsufficiency)
  • increased gene dosage
  • ectopic or temporally altered mRNA expression
  • increased or constitutive protein activity
  • dominant negative effects
  • altered structural proteins
  • toxic protein alterations
  • new protein functions

61
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 gt 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

64
Waardenburg Syndrome (PAX3)
  • Deafness
  • pigmentary anomalies
  • white forelock
  • heterochromia iridis
  • partial albinism,
  • Prominent broad nasal root
  • Hypertrichosis of the medial part of the eyebrows

65
heterochromia iridis
66
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 gtCharcot-Marie-Tooth disease
  • Haploinsufficient gt different phenotype of
    Increased Dosage!

67
Increased Dosage in Charcot-Marie-Tooth disease
68
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

69
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HPFH as a dß-globin Disease
  • Large deletions at the ß-globin locus
  • from the region close to the human A? gene to
    well downstream of the human ß-globin
  • gene and including deletion of the structural d-
    and ß-globin genes

72
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

73
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

74
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, b6GlugtVal)
  • Although recessive
  • Coinheritance in cis (hem S b23ValgtIle)
  • Sickling to manifest in the heterozygote!

75
Toxic protein alterations
  • Various point mutations in rhodopsin
  • Slow degeneration of rod photoreceptor outer
    segment

76
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 (922)
    Philadelphia translocation

77
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
78
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

79
Beckwith-wiedermann syndrome (BWS)
  • The incidence of BWS
  • 113700 live births
  • The increased risk of tumor formation in BWS
    patients
  • 7.5
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