Molecular pathology: Physiopathology effect of Mutations

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Molecular pathologyPhysiopathology effect of
Mutations

• Dr Derakhshandeh, PhD

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Mutations

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

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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

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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

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ClassificationBy effect on structure

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

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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

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Transversion

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

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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

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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

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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
  pseudo-chromosomes
• 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
  associated with speech and coordination
  difficulties

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• 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 !

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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

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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

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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

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Fibrillin gene
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Lethal mutations

• lead to a phenotype
• incapable of effective reproduction

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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

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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).

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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
  replace the wildtype

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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

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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 dont 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

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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
• 10-201 (RecDom)
• 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
  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

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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 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

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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 gtCharcot-Marie-Tooth disease
• Haploinsufficient gt 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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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

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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

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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!

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Toxic protein alterations

• Various point mutations in rhodopsin
• Slow degeneration of rod photoreceptor outer
  segment

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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

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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
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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
• 113700 live births
• The increased risk of tumor formation in BWS
  patients
• 7.5