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MUTATIONS OF THE DNA SEQUENCE
trisomy 21 (live birth), translocation trisomy 14 (early miscarriage), mono-
somy 14 or 21 (early miscarriage), a normal chromosome complement (live
birth), or a t(14q21q) carrier (live birth).
e.
A couple where one member is a t(14q21q) carrier may have a baby with
translocation trisomy 21 (Down syndrome) or recurrent miscarriages.
2.
Acute promyelocytic leukemia (APL) t(15;17)(q22;q21)
a.
APL t(15;17)(q22;q21) is caused by a reciprocal translocation between chro-
mosomes 15 and 17 with breakpoints at bands q22 and q21, respectively.
b.
This results in a fusion of the promyelocyte gene (PML gene) on 15q22 with
the retinoic acid receptor gene (RAR
gene) on 17q21, thereby forming the
PML/RAR
oncogene.
c.
The PML/RAR
oncoprotein (a transcription factor) blocks the differentiation
of promyelocytes to mature granulocytes such that there is continued prolif-
eration of promyelocytes.
d.
Clinical features include pancytopenia (i.e., anemia, neutropenia, and throm-
bocytopenia), including weakness and easy fatigue, infections of variable
severity, and/or hemorrhagic findings (e.g., gingival bleeding, ecchymoses,
epistaxis, or menorrhagia), and bleeding secondary to disseminated intravas-
cular coagulation. A rapid cytogenetic diagnosis of this leukemia is essential
for patient management because these patients are at an extremely high risk
for stroke.
3.
Chronic myeloid leukemia (CML) t(9;22)(q34;q11.2)
a.
CML t(9;22)(q34;q11.2) is caused by a reciprocal translocation between chro-
mosomes 9 and 22 with breakpoints at q34 and q11.2, respectively. The re-
sulting derivative chromosome 22 (der22) is referred to as the Philadelphia
chromosome.
b.
This results in a fusion of the ABL gene on 9q34 with the BCR gene on
22q11.1, thereby forming the ABL/BCR oncogene.
c.
The ABL/BCR oncoprotein (a tyrosine kinase) has enhanced tyrosine kinase
activity that transforms hematopoietic precursor cells.
d.
Clinical features include systemic symptoms (e.g., fatigue, malaise, weight
loss, excessive sweating), abdominal fullness, bleeding episodes due to platelet
dysfunction, abdominal pain may include left upper quadrant pain, early sati-
ety due to the enlarged spleen, tenderness over the lower sternum due to an
expanding bone marrow, and the uncontrolled production of maturing granu-
locytes, predominantly neutrophils, but also eosinophils and basophils.
G. UNSTABLE EXPANDING REPEAT MUTATIONS (DYNAMIC MUTATIONS; Fig-
ure 8-10). Dynamic mutations are mutations that involve the insertion of a repeat
sequence either outside or inside the gene. Dynamic mutations demonstrate a thresh-
old length. Below a certain threshold length, the repeat sequence is stable, does not
● Figure 8-10 Dynamic Mutation: Loss or Gain of Function.
Dynamic mutation
Loss of function or gain of function
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CHAPTER 8
cause disease, and is propagated to successive generations without change in length.
Above a certain threshold length, the repeat sequence is unstable, causes disease, and
is propagated to successive generations in expanding lengths. The exact mechanism by
which expansion of the repeat sequences occurs is not known. One of the hallmarks of
diseases caused by these mutations is anticipation which means the age of onset is
lower and degree of severity is worsened in successive generations. Dynamic mutations
are divided into two categories:
1.
Highly expanded repeats outside the gene.
In this category of dynamic muta-
tion, various repeat sequences (e.g., CGG, CCG, GAA, CTG, CCTG, ATTCT, or
CCCCGCCCCGCG) undergo very large expansions. Below threshold length ex-
pansions are
5–50 repeats. Above threshold length expansions are 100–1000
repeats. This category of dynamic mutations is characterized by the following clin-
ical conditions.
a.
Fragile X syndrome (Martin-Bell syndrome)
i. Fragile X syndrome is an X-linked recessive genetic disorder caused by a
200–1000
unstable repeat sequence of (CGG)
n
outside the FMR1 gene
on chromosome X for the fragile X mental retardation 1 protein
(FMRP1) which is a nucleocytoplasmic shuttling protein that binds sev-
eral mRNAs found abundantly in neurons.
ii. The 200–1000
unstable repeat sequence of (CGG)
n
creates a fragile site
on chromosome X which is observed when cells are cultured in a folate-
depleted medium. The 200–1000
unstable repeat sequence of (CGG)
n
has also been associated with hypermethylation of the FMR1 gene so that
FMRP1 is not expressed which may lead to the phenotype of fragile X.
iii. Fragile X syndrome involves two mutation sites. Fragile X site A involves
a 200–1000
unstable repeat sequence of (CGG)
n
located in a 5
UTR of
the FMR 1 gene on chromosome Xq27.3. Fragile X site B involves a 200
unstable repeat sequence of (CCG)
n
located in a promoter region of the
FMR 1 gene on chromosome Xq28.
iv. Normal FMR1 alleles have
5–40 repeats. They are stably transmitted
without any decrease or increase in repeat number.
v. Premutation FMR1 alleles have
59–200 repeats. They are not stably
transmitted. Females with permutation FMR1 alleles are at risk for hav-
ing children with fragile X syndrome.
vi. Clinical features include mental retardation (most severe in males),
macroorchidism (postpubertal), speech delay, behavioral problems (e.g.,
hyperactivity, attention deficit), prominent forehead and jaw, joint laxity,
and large dysmorphic ears. Fragile X syndrome is the second leading cause
of inherited mental retardation (Down syndrome is the number one
cause).
2.
Moderately expanded CAG repeats with the gene.
In this category of dynamic
mutation, a CAG repeat sequence undergoes moderate expansions. Below thresh-
old length expansions are
10–30 repeats. Above threshold length expansions are
40–200 repeats. Since CAG codes for the amino acid glutamine, a long tract of
glutamines (polyglutamine tracts) will be inserted into the amino acid sequence of
the protein and causes the protein to aggregate within certain cells. This category
of dynamic mutations is characterized by the following clinical conditions.
a.
Huntington disease (HD)
i. HD is an autosomal dominant genetic disorder caused by a 36 S 100
unstable repeat sequence of (CAG)
n
in the coding sequence of the HD
gene on chromosome 4p16.3 for the huntingtin protein which is a widely
expressed cytoplasmic protein present in neurons within the striatum,
cerebral cortex, and cerebellum although its precise function is unknown.
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MUTATIONS OF THE DNA SEQUENCE
ii. Since CAG codes for the amino acid glutamine, a long tract of glutamines
(a polyglutamine tract) will be inserted into the huntingtin protein and
cause protein aggregates to form within certain cells (such as implicated
in other neurodegenerative disorders).
iii. Normal HD alleles have
26 repeats. They are stably transmitted without
any decrease or increase in repeat number.
iv. Premutation HD alleles have 27–35 repeats. They are not stably transmit-
ted. Individuals with permutation HD alleles are at risk for having children
with HD. A child with HD inherits the expanded repeat from the father.
v. An inverse correlation exists between the number of CAG repeats and the
age of HD onset: 60–100 CAG repeats
juvenile onset of HD and 36–55
CAG repeats
adult onset of HD.
vi. Clinical features include the following: age of onset is 35–44 years of
age; mean survival time is 15–18 years after onset; a movement jerkiness
most apparent at movement termination; chorea (dance-like movements);
memory deficits; affective disturbances; personality changes; dementia;
diffuse and marked atrophy of the neostriatum due to cell death of cholin-
ergic neurons and GABAergic neurons within the striatum (caudate
nucleus and putamen) and a relative increase in dopaminergic neuron ac-
tivity; and neuronal intranuclear aggregates. The disorder is protracted
and invariably fatal. In HD, homozygotes are not more severely affected
by the disorder than heterozygotes, which is an exception in autosomal
dominant disorders.
Loss of Function and Gain of Function Mutations.
This is another way to classify
mutations that is commonly used.
A. LOSS OF FUNCTION MUTATION
1.
A loss of function mutation may be caused by a missense mutation (produces a
compensated protein), a nonsense mutation (produces unstable mRNAs or a non-
function truncated protein), a frameshift mutation (produces unstable mRNAs or
a nonfunctional garbled protein), RNA splicing mutation (produces unstable
mRNAs or a nonfunctional garbled protein), transposon mutation (produces no
protein), a translocation mutation (produces no protein), or a dynamic mutation.
Consequently, there are many ways to cause a loss of function mutation.
2.
For loss of function mutations to become clinically relevant, the individual needs
to be homozygous recessive (i.e., rr); heterozygotes (i.e., Rr) are clinically normal.
This is because for most genes, an individual can remain clinically normal by pro-
ducing only 50% of the gene product. This is why individuals with an inborn er-
ror of metabolism disease are homozygous recessive (rr).
3.
However, for a relatively few genes, an individual cannot remain clinically normal
by producing only 50% of the gene product (i.e., these genes show haploinsuffi-
ciency). Consequently, in haploinsufficiency, the 50% reduction in gene product
produces a clinically abnormal phenotype.
B. GAIN OF FUNCTION MUTATION
1.
A gain of function mutation may be caused by a missense mutation (produces a
compensated protein), a translocation mutation (produces a fusion protein with a
novel function; PML/RAR
oncoprotein or ABL/BCR oncoprotein), or a dynamic
mutation. Consequently, there are not many ways to cause a gain of function
mutation.
2.
For gain of function mutations to become clinically relevant, the individual needs
to be heterozygous (i.e., Rr). This is because the mutant allele (R) functions
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CHAPTER 8
abnormally despite the presence of a normal allele (r). A clinical example of a gain
of function mutation involves the Pittsburgh variant as follows:
a.
1
-Antitrypsin deficiency.
1
-Antitrypsin deficiency is an autosomal reces-
sive genetic disorder caused by a missense mutation in the SERPINAI gene
on chromosome 14q32.1 for the serpin peptidase inhibitor A1 (also called
1
-antitrypsin). In this missense mutation, methionine 358 is replaced with
arginine (i.e., the Pittsburgh variant) which destroys the affinity for elas-
tase. Methionine 358 at the reactive center of
1
-antitrypsin acts as a “bait”
for elastase where elastase is trapped and inactivated. This protects the phys-
iologically important elastic fibers present in the lung from destruction.
The Pittsburgh variant results in pulmonary emphysema because tissue-
destructive elastase is allowed to act in an uncontrolled manner in the lung.
In addition, the Pittsburgh variant results in bleeding disorder because the
Pittsburgh variant acts a potent inhibitor (gain of function) of the thrombin-
fibrinogen reaction.
To understand polymorphisms, a number of defi-
nitions must be clear. First, a gene is a hereditary factor that interacts with the environ-
ment to produce a trait. Second, an allele is an alternative version of a gene or DNA seg-
ment. Third, a locus is the location of a gene or DNA segment on a chromosome (because
human chromosomes are paired, humans have two alleles at each locus). Fourth, a poly-
morphism is the occurrence of two or more alleles at a specific locus in frequencies
greater than can be explained by mutations alone (a polymorphism does not cause a
genetic disease). Silent mutations may accumulate in the genome where they are called
single nucleotide polymorphisms. In addition, satellite DNA, minisatellite DNA, and
microsatellite DNA (all of which are tandemly repeated noncoding DNA) are prone to
deletion/insertion polymorphisms, whereby the number of copies of the tandem repeat
sequence varies. These are called variable number tandem repeat (VNTR) polymor-
phisms.
● Figure 8-11 Unequal Crossover.
Sister chromatid 1
Sister chromatid 2
Sister chromatid 1
Sister chromatid 2
Paternal
Unequal
crossover
Maternal
1
2
3
1
2
3
1
2
3
3
1
2
A. CAUSES OF VNTR POLYMORPHISMS.
VNTR polymorphisms can be caused in three
ways as follows:
1.
Unequal crossover (Figure 8-11).
Dur-
ing Meiosis I when crossover occurs, the
exchange of large segments of DNA be-
tween the maternal chromatid and pater-
nal chromatid (i.e., nonsister chro-
matids) at the chiasma is an equal
exchange, whereby the cleavage and re-
joining of the chromatids occurs at the
same position on the maternal chromatid
and paternal chromatid. In unequal
crossover, the cleavage and rejoining of
the chromatids occurs at different positions on the maternal chromatid and pa-
ternal chromatid (i.e., nonsister chromatids) usually within a region of tandem
repeats. This diagram shows an example of unequal crossover. A polymorphism
results in the maternal chromatid having an extra repeat sequence (no. 3)
obtained from the paternal chromatid (nos. 1, 2, and 3
copies of the tandem
repeat sequence).
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MUTATIONS OF THE DNA SEQUENCE
2.
Unequal sister chromatid exchange
(UESCE; Figure 8-12).
During Meiosis I
when crossover occurs, the cleavage and
rejoining of sister chromatids occurs at
different positions on the maternal chro-
mosome usually within a region of tan-
dem repeats. Or the cleavage and rejoin-
ing of sister chromatids occurs at different
positions on the paternal chromosome
usually within a region of tandem re-
peats. This diagram shows an example of
● Figure 8-12 Unequal Sister Chromatid
Exchange.
Sister chromatid 1
Sister chromatid 2
Sister chromatid 1
Sister chromatid 2
Paternal
UESCE
Maternal
1
2
3
1
2
3
1
3
1
2
2
3
● Figure 8-13 Replication Slippage.
strand has no. 4 repeat sequence deleted. An insertion polymorphism occurs due
to backward slippage so that the newly synthesized DNA strand has an extra no. 2
repeat sequence inserted.
B. TYPES OF VNTR POLYMORPHISMS
1.
Large-scale VNTR polymorphisms.
Large-scale VNTR polymorphisms are typically
found in satellite DNA which is composed of very large sized blocks (100 kb S
several Mb) of tandem-repeated noncoding DNA and are formed by both unequal
crossover and UESCE.
2.
Simple VNTR polymorphisms.
There are two types which include
a.
Minisatellite DNA polymorphisms. Minisatellite DNA polymorphisms are
typically found in minisatellite DNA which is composed of moderately sized
blocks (0.1 kb S 20 kb) of tandem repeated noncoding DNA and are formed
by replication slippage.
b.
Microsatellite DNA or SSR (simple sequence repeat) polymorphisms.
Microsatellite DNA or SSR polymorphisms are typically found in microsatel-
lite DNA which is composed of small-sized blocks (1–6 bp) of tandem-repeated
noncoding DNA and are formed by replication slippage.
UESCE. A polymorphism results in one sister maternal chromatid having two
repeat sequences (no. 1 and no. 3) and the other sister maternal chromatid having
four repeat sequences (nos. 1, 2, 2, and 3).
3.
Replication slippage (Figure 8-13).
Dur-
ing Meiosis I when DNA replication oc-
curs, a region of tandem repeats does not
pair faithfully with the region of tandem
repeats on its complementary strand. If the
DNA loop forms on the template strand, a
forward slippage occurs and causes a dele-
tion polymorphism. If the DNA loop
forms on the nascent strand, a backward
slippage occurs and causes an insertion
polymorphism. This diagram shows ex-
amples of replication slippage. A deletion
polymorphism occurs due to forward slip-
page so that the newly synthesized DNA
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