12. Molecular Basis of Genetic Diseases

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12. Molecular Basis of Genetic Diseases
a). Determinants of phenotypic expression
i). Nature of the mutation ii). Genetic backg
round iii). Environmental influences b). Type
s of mutations affecting genes
i). Regulatory mutations factor IX promoter
mutations, etc. ii). Mutations affecting the pr
otein product b-globin mutations iii). Larg
e-scale gene mutations b-globin deletions
dystrophin deletions iv). Trinucleotide repeat
expansion mutations neuropsychiatric disorders

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• Learning objectives
• know the determinants of phenotypic expression
• understand the concept that different mutations
  in the same gene
  
• can have profoundly different phenotypic effects
• know the mechanisms by which different point
  mutations in genes can
  
• affect regulatory processes, e.g. transcription,
  translation, etc.
  
• understand the concept that two different
  mutations in the b-globin
  
• gene that affect the same amino acid can have
  significantly
  
• different phenotypes
• understand how unequal crossing over and gene
  deletion can
  
• give rise to thalassemias of vastly different
  severity
  
• understand how small and large deletions in the
  dystrophin gene
  
• can give rise to more and less severe forms of
  MD, respectively
  
• understand the molecular basis for the
  neuropsychiatric diseases
  
• caused by trinucleotide repeat expansion
• understand the terms premutation and
  anticipation
  
• understand the concept that disease severity can
  depend on the
  
• number of expanded triple repeats

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• Determinants of phenotypic expression
• Nature of the mutation
• different mutations in the same gene can lead to
  more- or
  
• less-severe phenotypes depending on the
  effects of the
  
• mutations on the expression of the gene or
  on the function
  
• of the protein product
• Genetic background
• two individuals within a family may have the
  same mutated gene,
  
• however, they will certainly (unless they
  are identical twins) have
  
• a lot of genes that are not similar -- the
  expression of these
  
• background genes may influence the disease
  phenotype
  
• Environmental influences
• factors such as lifestyle, diet, and exposure to
  environmental
  
• toxins may affect the disease phenotype

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• Nature of the mutation
• regulatory mutations
• point mutations in the Factor IX promoter
• mutations affecting the protein product
• point mutations in the b-globin gene
• large-scale gene mutations
• deletion mutations of the globin genes
• deletion mutations in the dystrophin gene
• trinucleotide repeat expansion mutations
• neuropsychiatric disorders

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Regulatory mutations

• The factor IX gene
• located on the X chromosome
• transcribed region 32,700 bp, with 8 exons

• The factor IX gene promoter
• there are overlapping binding sites for AR and
  HNF4
  
• AR androgen receptor
• zinc finger nuclear receptor superfamily
  transcription factor
  
• binds androgen
• androgen levels increase at puberty
• HNF4 hepatocyte nuclear factor-4
• zinc finger nuclear receptor superfamily
  transcription factor
  
• ligand unknown - therefore an orphan receptor
• HNF4 is expressed early in development and in
  adult liver

-27
-15
-36
-22
HNF4
AR
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• mutation at -20 results in
• Hemophilia B Leyden in which
• the hemophilia improves at puberty
• when levels of androgen increase

-27
-15
-36
-22
HNF4
AR

• mutation at -26 results in
• Hemophilia B Brandenburg
• in which factor IX levels remain low even after
  puberty

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Regulatory mutations affecting either the 5' UTR
or the 3' UTR
Iron-responsive element (IRE)
5 UTR
3 UTR
5
3
AUG
UGA
Translated region (open reading frame)
Internal ribosome entry site (IRES)
Hereditary thrombophilia
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Regulatory mutations affecting either the 5' UTR
or the 3' UTR 1). Some mRNAs recruit ribosomes
directly to initiating AUG codons through 5'
untranslated region (UTR) elements termed
internal ribosome entry sites (IRESs). Mutation
of the IRES in the connexin-32 gene is a cause of
Charcot-Marie-Tooth disease. Mutation of the IRES
of the c-myc gene causes increased translation of
this oncogene, leading to multiple myeloma
(Mendell and Dietz, Cell 107411 2001).
2). The 5' untranslated regions (5' UTRs) of so
me mRNAs are known to regulate their translation.
Hyperferritinemia/ cataract syndrome (HHCS) can
be caused by mutations that affect an
iron-responsive element (IRE) in the 5' UTR of
the L-ferritin gene, which functions in sensing
cellular iron levels. Mutation of this IRE
element in the 5' UTR results in abnormally high
L-ferritin production (Mendell and Dietz, Cell
107411 2001). 3). Hereditary thrombophilia (
increased tendency for the blood to clot) is
caused by a single nucleotide substitution in the
3' UTR, which is present in 1-2 of the
population. This mutation increases the
efficiency of 3 end processing, leading to
excess production of thrombin mRNA and protein
(Mendell and Dietz, Cell 107411 2001).
Pulmonary embolism is the most common cause, in
the industrialized world, of maternal death
during pregnancy or in the period following
delivery. About 70 of women who present with
venous thromboembolism during pregnancy are
carriers of hereditary or acquired thrombophilia
(Eldor, J Thromb Thrombolysis 1223 2001).
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• Developmental expression of the globin chains
• embryonic hemoglobins
• z2e2
• a2e2
• z2g2
• fetal hemoglobins
• a2g2 HbF
• adult hemoglobins
• a2d2 HbA2
• a2b2 HbA

• the arrangement of the a-like genes and
• the b-like genes reflects their order of
• developmental expression

z
a2
a1
e
Gg
Ag
d
b
developmental time from embryo to adult
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• Genetic disorders of hemoglobin
• structural variants in the proteins
  (hemoglobinopathies)
  
• sickle cell disease
• affects codon 6 of b-globin
• GAG (glutamic acid) to GTG (valine)
• results in a severe sickling disease
• hemoglobin C disease
• affects codon 6 of b-globin
• GAG (glutamic acid) to AAG (lysine)
• results in a mild hemolytic anemia
• thalassemias (decreased a- or b-chains)
• hereditary persistence of fetal hemoglobin
  (HPFH)
  

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• unequal crossing over
• a-thalassemia

• the two a-globin genes arose by gene
  duplication they are identical
  
• unequal crossing over gave rise to the loss of
  one of the genes
  
• inheritance of the deleted chromosome gives rise
  to a-thalassemia

a2
a1
X
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• normally there are four a-globin genes in
  heterozygotic somatic cells
  
• loss of a-globin genes results in different
  severities of a-thalassemia
  
• depending on the number of genes lost in
  combination with
  
• deletion chromosomes

a2
a1
normal
a2-thalassemia (silent carrier state)
a1-thalassemia (no significant anemia)
Hb H disease (mild to severe anemia)
hydrops fetalis (fetal or early neonatal death)
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• unequal crossing over
• hemoglobin Lepore (b-thalassemia)

Gg
Ag
d
b
X
Hb Lepore
db-fusion

• unequal crossing over occurred due to the close
  homology
  
• of the d- and b-genes only 10 out of 146
  residues differ
  
• (the genes are 90 homologous to each other)
• the consequence can be severe b-thalassemia due
  to decreased
  
• synthesis of the db-fusion (due to the weak
  d-globin promoter)

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• large deletions (examples of large deletions
  with little or no phenotype)
  
• db-thalassemia -- some compensation by g-chain
  synthesis
  
• HPFH -- entirely compensated by g-chain synthesis

Gg
Ag
d
b
db-thalassemia
HPFH
(deleted regions are indicated by the black boxes
below the chromosome)
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• Mutations in the dystrophin gene
• Becker and Duchenne muscular dystrophy
• BMD is a less-severe disease (patients are still
  walking after 16 yrs)
  
• DMD is a more-severe disease (patients are not
  walking at 12 yrs)
  
• both can be caused by massive deletions in the
  dystrophin gene (as well
  
• as other types of mutations)
• the severity is not necessarily correlated with
  the size of the deletion

dystrophin cDNA (coding region)
dystrophin protein
spectrin-like repeat domain
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mutations causing BMD can be very large in-frame
deletions
truncated but functional protein with intact N-
and C-termini
partially functional dystrophin protein
mutations causing DMD can be small out-of-frame
deletions
C-terminal truncated protein (with out-of-frame
translation product)
non-functional dystrophin protein
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• Neuropsychiatric diseases caused by expansion
• of trinucleotide repeats
• Myotonic dystrophy
• Fragile X syndrome
• Spinal and bulbar muscular atrophy (Kennedys)
• Huntingtons disease

• Microsatellites
• short regions of repeating DNA sequence in the
  genome
  
• (because their GC content is usually higher or
  lower
  
• than the average for the genome they frequently
  appear
  
• to band at a different buoyant density in CsCl
  gradients
  
• and hence are called satellites)
• microsatellites are often comprised of
  trinucleotide repeats

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• Trinucleotide (or triple) repeats (a form of
  microsatellites)
  
• the most common in human DNA are
• CAG, CGG, CAA, TAA, GAG
• because DNA is double-stranded, the CAG repeat
  includes
  
• CAG, AGC, GCA, CTG, TGC, GCT
• 5CAGCAGCAGCAGCAG 3
• 3GTCGTCGTCGTCGTC 5
• the number of repeats, and therefore their
  length at any
  
• given locus, are polymorphic in the human
  genome
  
• giving rise to VNTRs (variable number of tandem
  repeats)

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Polymorphism at a triple repeat (VNTR) locus
CAG CAG CAG CAG CAG
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• Myotonic dystrophy
• autosomal dominant disease
• characterized by myotonia and progressive muscle
  weakness
  
• the most common form of adult-onset muscular
  dystrophy
  
• skeletal, cardiovascular, and ocular (cataracts)
  manifestations
  
• associated with cognitive changes, including
  mental retardation
  
• disease shows anticipation
• late onset of mild symptoms in the first
  generation to neonatal
  
• onset (only inherited from the mother) associated
  with mental
  
• retardation in a later generation (within 3-4
  generations)
  
• caused by mutations in the DM-1 or myotonin
  protein kinase gene
  
• which is expressed in brain, heart, and muscle
• normal gene has a 3-UTR CTG repeat that is
  polymorphic in the
  
• population and that ranges from 5-30 repeats
• patients have expanded numbers of repeats, up to
  many hundreds,
  
• that lead to decreased mRNA levels

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(No Transcript)
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• Anticipation
• severity increases over several generations
• mild symptoms in the first generation to severe
• symptoms in later generations
• due to stepwise expansion of unstable triple
  repeats
  
• in the normal population, the length of the
  repeat is
  
• polymorphic, but stable
• the first step is the formation of a
  premutation that has a
  
• normal phenotype but is unstable
• the premutation then expands in a subsequent
  generation to
  
• a much greater length and further instability
• anticipation is a hallmark of trinucleotide
  repeat expansion

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Structure and inheritance of CTG repeats
in myotonic dystrophy
affected 75
premutation 45-75
normal 5-30
myotonic protein kinase gene
(CTG)n
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• Fragile X syndrome (FRAXA)
• X-linked
• occurs in one of 1,250 male births and is the
  second most common
  
• cause of mental retardation
• associated with moderate to severe mental
  retardation often with
  
• developmental delays and autistic behavior
• disease shows anticipation
• increasing penetrance in succeeding
  generations
  
• passage through female can increase risk to next
  generation
  
• females with one affected chromosome and males
  with premutations
  
• can show mild cognitive defects and schizotypal
  symptoms
  
• caused by mutations in the FMR-1 gene which is
  expressed in
  
• brain and testes at highest levels, and widely in
  the embryo
  
• normal gene has a 5-UTR CGG repeat that is
  polymorphic in the
  
• population and that ranges from 6-55 repeats
• patients have expanded numbers of repeats, up to
  thousands
  
• results in transcriptional silencing

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• Penetrance and expressivity
• penetrance
• an all-or-nothing phenomenon that refers to the
• observable expression, or lack thereof
• a measure of the proportion of individuals with
  a particular
  
• gene (allele) that shows the phenotype for that
  gene
  
• expressivity
• the variation in phenotype associated with a
  particular allele
  
• due to genetic background or the environment
• most autosomal dominant conditions show
• incomplete penetrance, and
• variable expressivity

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Structure and inheritance of CGG repeats
in fragile X syndrome
affected 200
premutation 55-200
normal 6-55
FMR-1 gene
(CGG)n
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• Spinal and bulbar muscular atrophy (Kennedys)
• X-linked rare
• late onset form of motor neuron degeneration
  associated with
  
• mental retardation and insensitivity to
  androgens
  
• caused by mutations in the androgen receptor
  gene
  
• normal gene has a CAG repeat that is polymorphic
  in the population
  
• and that ranges from 13-28 repeats
• patients have expanded numbers of repeats, up to
  39-60 repeats
  
• CAG repeat is translated into a polyglutamine
  tract in the protein
  
• disease shows anticipation, but the severity of
  the illness does not
  
• correlate with the degree of expansion

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Structure and inheritance of CAG repeats
in spinal and bulbar muscular atrophy
affected 39-60
normal 13-28
androgen receptor gene
(CAG)n (Gln)n
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• Huntingtons disease
• autosomal dominant disease
• juvenile to late adult onset
• associated with involuntary movements (chorea),
  behavioral
  
• disturbances, and cognitive impairment
• caused by mutations in the IT15 gene (whose
  function is unknown)
  
• normal gene has a CAG repeat that is polymorphic
  in the population
  
• and that ranges from 11-35 repeats
• patients have expanded numbers of repeats (35
  repeats)
  
• CAG repeat is translated into a polyglutamine
  tract in the protein
  
• disease shows anticipation, but the severity of
  the illness does not
  
• always correlate strictly with the degree of
  expansion
  
• affected children of affected fathers have an
  age of onset 8-10
  
• years earlier than their fathers
• affected children of affected mothers have an
  age of onset
  
• similar to their mothers

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CAG repeats in Huntingtons disease
affected range 39 repeats
may or may not have disease 36-39 repeats
normal but can expand 27-35 repeats
normal range 11-35 repeats
(CAG)n (Gln)n
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• Other triple repeat diseases
• spinocerebellar ataxia type 1 (SCA1) (CAG
  polyglutamine)
  
• dentatorubral-pallidoluysian (DRPLA) (CAG
  polyglutamine)
  
• Machado-Josephs disease (MJD/SCA3) (CAG
  polyglutamine)
  
• FRAXE mental retardation (GCC)
• Jacobsons disease (GCC)
• Other diseases showing anticipation
• bipolar affective disorder (possible CAG
  expansion)
  
• schizophrenia (possible CAG expansion)
• autism not associated with fragile X syndrome
• spastic paraplegia
• hereditary Parkinsons disease

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• Conclusions (triple repeat diseases)
• expansion of trinucleotide repeats represent
  dynamic mutations
  
• trinucleotide repeat expansions cause unusual
  forms of
  
• inheritance, including anticipation
• a number of neuropsychiatric disorders,
  including bipolar affective
  
• disorder and schizophrenia show anticipation
• repeat disorders can be classified into two
  distinct types in which
  
• the repeats are translated (type I) or not
  translated (type II)
  
• a number of novel genes containing relatively
  long, polymorphic
  
• trinucleotide repeats are expressed in the human
  brain,
  
• which may be candidates for other
  neuropsychiatric disorders