Download Prof. Kamakaka`s Lecture 11 Notes

Mutations
1
What is a gene?
Prokaryotic Genes
PROMOTER
3’
5’
antisense
---TTGACAT------TATAAT-------AT-/-AGGAGGT-/-ATG CCC CTT TTG TGA
---AACTGTA------ATATTA-------TA-/-TCCTCCA-/-TAC GGG GAA AAC ATT
sense
3’
(-35)
(-10)
5’
RIBOSOME
BINDING
SITE
3’
5’
U-/-AGGAGGU-/-AUG CCC CUU UUG UGA
Met Pro leu leu stp
When all of these rules are satisfied then A segment of DNA will
generate an RNA which will then be read by a ribosome and be
translated into a protein.
2
Reading the genetic code
5’
3’
A T G T T T A A A T A G C C C
G G G C T A T T T A A A C A T
3’
5’
5’
3’
A T G T T T A A A T A G C C C
5’
3’
A U G U U U A A A U A G C C C
G G G C T A T T T A A A C A T
3’
5’
5’
3’
A U G U U U A A A U A G C C C
U A C A A A U U U S T P
3
Met
Phe
Lys
No Gaps
5’
3’
A U G U U U A A A U A G C C C
U A C A A A U U U S T P
Met
Phe
Lys
5’
3’
A U G U U U A A A U A G C C C
U A C
A A U U U A
Met
Asn
Leu
4
No overlaps
5’
3’
A U G A A A C C C U A G C C C
U A C U U U G G G S T P
Met
Lys
Pro
5’
3’
A U G A A A C C C U A G C C C
U A C U U U G G
Met
Lys Trp
5
The GENETIC CODE
The code is a three letter code.
Second letter
U
C
CUU
CUC
CUA
CUG
A
AUU
AUC
AUA
AUG
G
GUU
GUC
GUA
GUG
Phe
Leu
Leu
Ile
Met
Val
A
UCU
UCC
UCA
UCG
UAU
UAC
UAA
UAG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Ser
G
Tyr
STOP
His
Pro
CAU
CAC
CAA
CAG
Asn
Thr
AAU
AAC
AAA
AAG
Ala
GAU
GAC
GAA
GAG
Gln
Lys
Asp
Glu
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG
U
C
A
G
Cys
STOP
Trp
U
C
A
G
Arg
U
C
A
G
Ser
Arg
U
C
A
G
Gly
6
Third letter
First letter
U
UUU
UUC
UUA
UUG
C
The code
3
5
1
9
2
amino
amino
amino
amino
amino
acids are specified by 6 different codons
acids are specified by 4 different codons
acid is specified by 3 different codons
acids are specified by 2 different codons
acids are specified by 1 different codons
The degeneracy arises because
More than one tRNA specifies a given amino acid
A single tRNA can base-pair with more than one codon
tRNAs do not normally pair with STOP codons
Ser
Ser
Ser
AGG
AGU
UCG
----UCC------UCA------AGC
Ser
Ser
AGG
AGG
----UCC------UCA------
7
The Genetic Code
Properties of the Genetic code:
1- The code is written in a linear form using the nucleotides
that comprise the mRNA
2- The code is a triplet: THREE nucleotides specify
ONE amino acid
3- The code is degenerate: more than one triplet specifies
a given amino acid
4- The code is unambiguous: each triplet specifies only
ONE amino acid
5- The code contains stop signs- There are three different stops
6- The code is comma less
7- The code is non-overlapping
8
xxxxxx
9
Mutations
Most mutations are harmful in their effects; only rarely are
mutations beneficial.
A gene with one wild-type allele is monomorphic; a gene with
two or more wild-type alleles is polymorphic.
The vast majority of traits are determined by alleles of more
than one gene. This means that most traits are multifactorial
traits.
A Heterogeneous Trait is one that may be caused by mutations
in more than one gene.
Human deafness is an example of a heterogeneous trait:
Mutations in any of at least 50 genes lead to deafness.
An important class of mutations are conditional mutations(Environment affects Phenotype). Conditional mutations are
those that express their associated phenotype only under some
conditions (restrictive conditions) and not others (permissive
conditions).
Conditional lethal mutations are common.
Temperature-sensitive conditional mutations are invaluable in
genetic research.
10
Generation of mutations
Spontaneous mutations
Replication induced mutations of DNA
Usually base substitutions (Most errors are corrected)
Meiosis- segregation defects or defects during
crossing over can induce mutations
Small additions and deletions AND Large changes as well
Environment induced changes
Exposure to physical mutagens - Radioactivity or chemicals
Depurination (removal of A or G)
Repair results in random substitution during replication
Deamination (removal of amino group of base)
Cytosine--uracil--bp adenine--replication--
(nitrous acid)
Oxidation (oxoG)
guanine--oxoguanine--bp adenine--replication -Base analog incorporation during replication BU-T
Intercalating agents
11
Mutation rate
There are approximately 1013 cells in the human body
Each cell receives 1-10,000 DNA lesions per day (Lindahl and
Barnes 2000). Almost all are repaired!!
Most pervasive mutagen is UV. 100,000 lesions per exposed
cell per hour (Jackson and Bartek 2009).
Ionizing agents (X-rays/g-rays) are most toxic because they
generate double strand breaks (Ward 1988).
Chromosome instability (gain or loss of entire segments) is
frequent - 40% of imbalances are entire arm imbalance while
45% are terminal segment imbalance (double strand break, nondysjunction etc)
Sequencing 179 humans as part of the 1000 genome project:
On average, each person is found to carry approximately 250
to 300 loss-of-function variants in genes of which 50 are in
genes previously implicated in genetic disorders.
Each individual on average has:
1.3 million short indels (1-10,000 bp) and 20,000 large
sequence variations (>10,000 bp).
Variation detected by the project is not evenly distributed
across the genome: certain regions, containing repetitive
sequences (sub-telomeres etc), show high rates of indels.
12
Sequencing the whole genomes of a family (2010 Science 328
636).
98 crossovers in maternal genome
57 crossovers in paternal genome
Mutation rate is 1x10-8 per position per haploid genome
(human genome is 3x109 bp)
It was calculated that there are ~70 new mutations in each
diploid human genome
Some sites such as CpG sites mutate at a rate 11 times
higher than other sites
Exome sequencing of 2440 individuals (Science 2012 337 40)
Each person has ~100 loss of function mutations (~35
nonsense).
20 loss of function mutations are homozygous
Some alterations in sequence concentrate in specific
geographic populations.
Rare changes are population specific and their frequencies
vary for each geographic population
13
Methods used to study mutations
Gross chromosomal changesdeletions, insertions, inversions, translocations
Cytology- microscopy- karyotype
Small mutations
Small deletions, insertions and point mutations
Recombinant DNA technologies
14
Chromosomes and chromosome rearrangements
Cytogenetics is the study of genetics by visualizing
chromosomes. This area of research is germane to several
areas of biological research.
Cytogenetics has been fundamental to understanding the
evolutionary history of a species (for example, although the
Chimp and the human are morphologically very different, at the
level of the chromosome (and DNA sequence) they are
extremely similar.
H = human
C= chimp
G = Gorilla
O = Orang utang
15
Karyotype
Chromosomes are classified by size, centromere position and
banding pattern:
Shown below is the human karyotype (description of the
chromosome content of a given species)
Karyotype is the chromosome description of length, number,
morphology.
Karyotype analysis is extremely important in medicine.
Alternations in karyotypes are linked to birth defects and many
human cancers.
Metacentric- centromere in the middle
Acrocentric- centromere off center
telocentric centromere at one end
16
Banding patterns
Specialized stains produce unique banding patterns along each
chromosome. Banding patterns are extremely useful for
detecting abnormalities in chromosome structure.
For many of the chromosome stains- the molecular basis of
the banding patterns is unclear. Nonetheless these techniques
remain fundamental in many areas of genetic research
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MU to bp
Genetic maps are based on recombination frequencies and
describe the relative order and relative distance between
linked genes.
Remember genes reside on chromosomes.
So what we would like to know is where are the genes located
on the chromosomes
What does this mean in terms of chromosomes and DNA?
18
Physical maps
Physical maps provide information concerning the location of
genes on chromosomes
Where are the genes on chromosomes?
Cytological studies have been successfully used to map genes to
specific regions of a chromosome.
For example in Drosophila in some cells the chromosomes
become highly replicated and exhibit very characteristic
banding patterns:
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In situ hybridization
Salivary glands
Squash on slide
Denature/Stain polytene chromosomes
label gene probe (you can only use this method if you have the
gene cloned)
Hybridize probe to polytene chromosomes
Autoradiography
20
Chromosome loss/gain
Chromosome instabilityElevated gain or loss of complete chromosomes
Very Frequent in tumors
Frequent in in vitro fertilized embryos
Increase with age of parent
Males have 2% rate of aneuploidy during gamete formation
Females have 15% rate of aneuploidy (that increases to 60% by age
50)
(25% of all conceptions result in miscarriage due to aneuploidy usually at implantation
stage)
Gross chromosomal rearrangements during in vitro fertilization.
40% of embryos carried entire chromosome imbalance
Entire chromosome aneuploidy in human adults:
Only chromosome 13, 18, 21 and X/Y
21
Gross chromosomal changes
The Cri du chat syndrome in humans is a result of a deletion in
the short arm of chromosome 5. This was determined by
comparing banding patterns with normal and Cri du Chat
individuals
Types of chromosome rearrangements that can be studied by
karyotype analysis:
GROSS CHROMOSOMAL CHANGES
Deletions, Duplications, Inversions, Translocations
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Copy Number Variation of chromosomes
CNV- copy number variation of entire chromosomes or
of chromosome segments
5% of an individuals genome displays CNV
Synuclein gene (involved in membrane stability of
neurons). CNV is involved in Parkinsons
Many cancers- malignant cells most often gain additional
copies of chromosome segments- genes in these
segments are mis-expressed and this leads to misexpression of other genes.
Tissues of identical twins show copy number differences
at several loci (Bruder et al 2008)
Different tissues of single individual show copy number
differences (Piotrowski et al., 2008)
Gross chromosomal rearrangements during in vitro
fertilization
55% of embryos carried terminal imbalance (subtelomere loss) (Vanneste et al., 2009 -microarray based
screen of IVF 35 embryo)
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DDIT
Normal Chromosome
A____B____C________D____E____F
Deletions (deficiency)
A____B____C________D____F
Duplications
A____B____C________D____E____E____F
Inversions
A____B____C________E____D____F
Translocation
A____B____C________D____E____F
A____B____C________D____L
H____I____J________K____L
24
H____I____J________K____E____F
Insertion and deletions are frequent: Sequencing 179 humans
as part of the 1000 genome project:
On average, each person is found to carry approximately
250 to 300 loss-of-function variants in genes of which 50
are in genes previously implicated in inherited disorders.
20,000 large structural variants were identified and 1.3
million short indels were identified.
Variation detected by the project is not evenly distributed
across the genome: certain regions, such as subtelomeric
regions, show high rates of variation.
1 in 500 children have reciprocal translocation but such
translocations are usually harmless. (However gametes
produced by the children will have defects).
1 in 50 children have inversions (small and large). The
heterozygous inversion carrier generally show no adverse
phenotype (but produce abnormal meiotic products from
crossing-over in the inversion loop).
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Deletions
Deletions are often detected cytologically by comparing
banding patterns between the normal and the partially
deleted chromosomes
Deleted
segment
Chromosome no
female
deletion
chromosome1
Band
46,XX, del(1)(q24q31)
Female with a deletion of chromosome 1 on the long arm (q)
between bands q24 to q31.
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In many instances deletions are too small to be detected
cytologically. In these instances genetic/molecular techniques
are used.
Since cytological deletions remove a contiguous set of genes,
there is a high probability that an essential gene will be
deleted. Therefore deletions will survive as heterozygotes
and not homozygotes.
A____B________C____D
Normal
A____B________C____D
A____________C____D
A____________C____D
A____________C____D
A____B________C____D
Homologous deletion
(Lethal?)
Heterologous deletion
(NOT Lethal)
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Consequences of deletions
A+_____B+_____C+___________D+
Normal
A+_____B+_____C+___________D+
B+
A+____/ \_____C+___________D+
A+___________C+___________D+
In individuals heterozygous
for the deletion, pairing is
disrupted in the regions
surrounding the deletion.
Therefore recombination is
also significantly reduced in
these regions.
Genotype
A+_____b______c____________D+
Normal
A+_____B+_____C+___________D+
A+____b______c____________D+
A+___
_____C+___________D+
A deletion on one
homologue unmasks
recessive alleles on the
other homologue. The
effect is called pseudo28
dominance.
Deletions in X
Females in Drosophila
XX
Males in Drosophila
XY or XO
Deletion series
phenotype
sick
dead
sick
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Changes in chromosome structure
Deletions:
1.
Hemizygosity from large deletions results in lethalityeven the smallest cytologically defined deletions take out
tens of 1,000's of bps and are likely to remove essential
genes.
2. Organisms can tolerate hemizygosity from small but not
large deletions. The reason for this is not entirely clear
and is placed under the rubric of disrupting the overall
ratio of gene products produced by the organism
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xxxxxxxx
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Duplications
A____B____C________D____E____F
A____B____C________D____E____E____F
normal
Duplication
Individuals bearing a duplication possess three copies of the genes
present in the duplicated region.
In general, for a given chromosomal region, organisms tolerate
duplications much better than deletions.
46,XY, dup(7)(q11.2q22)
Male with a duplication of chromosome 7 on the long arm (q) 32
between bands 11.2 to 22
Tandem Duplications
Tandem duplications- Important class of duplications!!!
This is a case in which the duplicated segment lies adjacent to
the original chromosomal segment
A B C D E ------ A B C B C B C B C D E
Once a tandem duplication arises in a population, even more
copies may arise because of asymmetrical pairing at meiosis.
Remember when the homologs pair during prophase of meiosis
I, they line up base-pair for base pair. Duplications lead to
mistakes in this pairing mechanism
33
Pairing of duplicated segments
Proper pairing:
A____B____C____B____C____D____E
A____B____C____B____C____D____E
A____B____C____B____C____D____E
A____B____C____B____C____D____E
Inappropriate pairing:
A____B____C____B____C____D____E
A____B____C____B____C____D____E
A____B____C____B____C__-----------__D____E
A____B____C____B____C__-----------__D____E
34
Pairing of duplicated segments
Tandem duplications expand by mistakes in meiosisI during
pairing
A
b
B
c
C
d
D
e
E
B
a
C
A
B
C
D
E
b
a
b
c
d
e
c
Sister chromatids
Sister chromatids
35
Normal pairing of chromosomes
pairing of chromosomes in repetitive regions
Pairing of duplicated segments
Pairing of duplicated segments
Tandem duplications expand by mistakes in meiosis during
pairing
Paired non-sister chromatids
B
C
A
B
C
D
E
a
b
c
d
e
b
c
37
Crossover in mispaired duplicated segments
What happens if you get a crossover after mis-pairing in
meiosisI?
A
B
C
B
C
D
A
B
C
B
C
D
b
c
b
c
a
b
a
B
c
b
c
d
d
C
A
B
C
D
E
a
b
c
d
e
b
c
A
B
C
B
C
D
A
B
C
B
C
B
A
B
C
D
A
B
C
B
C
D
C
D
38
The four meiotic products of a crossover between regions B
and C:
A-B-C-B-C-D-E
A-B-C-D-E
A-B-C-B-C-B-C-D-E
A-B-C-B-C-D-E
This process may repeat itself many times, such that a small
fragment of the genome is repeated 10,000 times.
39
An example of this is near the centromeres of the Drosophila
genome:
If you look at the DNA sequence in this region it consists of
small 5-10 bp sequences (AATAC)n repeated 1,000s of times.
It is believed to have arisen from unequal crossing over.
Repetitive DNA- cell does not like it- They try to reduce
recombination of repetitive DNA by packaging the DNA with
proteins to form heterochromatin- cold spots of recombination
along the chromosome
40
Duplications provide additional genetic material capable of
evolving new function. For example in the above situation if
the duplication for the B and C genes becomes fixed in the
population- the additional copies of B and C are free to evolve
new or modified functions.
This is one explanation for the origin of the tandemly
repeated globin genes in humans. Each of these has a unique
developmental expression pattern and provides a specialized
function.
The hemoglobin in fetus has a higher affinity for oxygen since
it acquires its oxygen from maternal hemoglobin via
competition
41
xxxxxx
42
Inversion
Chromosomes in which two breaks occur and the resulting
fragment is rotated 180 degrees and reinserted into the
chromosome.
Inversions involve no change in the amount of genetic material
when they occur and therefore they are often genetically viable
and show no abnormalities at the phenotypic level.
Gene fusions may occur
Inversions are defined as to whether they span the centromere
Paracentric inversions do not span the centromere:
A
B
A
C
B
D
D
E
C
E
Pericentric inversions span the centromere:
A
C
B
D
E
In a pericentric inversion one break is in the short arm and one in the
long arm. Therefore an example might read 46,XY,inv(3)(p23q27).
43
A paracenteric inversion does not include the centromere and an
example might be 46,XY,inv(1)(p12p31).
Homologs which are heterozygous for an inversion have
difficulties pairing in meiosis.
During pairing homologous regions associate with one another.
Consequently individuals heterozygous for an inversion will form
a structure known as an inversion loop.
Crossover within inverted region?
A---B---C----D---E----F----G
A’--B’---C’---D’--E’---F’---G’
A---B---C----D----E---F---G
A’--B’---C’---E’---D’--F’---G’
A
B
C
A’
B’
C’
D
E
D’
E’
F
G
‘F
G’
44
The consequence of crossover within a paracentric inversion
a-b-c
d-e
a-b-c
a-b-c
f-g
d-e
e-d
f-g
f-g
During meiosis, pairing leads to formation of an inversion loop
This is a problem if crossing over occurs within the inversion
A
B
C
A’
B’
C’
D
E
D’
E’
F
G
‘F
G’
A-B-0-C-D-E’-C’--0--B’-A’
dicentric-fragmentation
G-F-E-D’-F’-G’
acentric- no segregation
45
The consequence of crossover within a pericentric inversion
(one that spans the centromere).
a-b-c
d-e
a-b-c
a-b-c
f-g
d-e
e-d
f-g
f-g
During meiosis, pairing leads to formation of an inversion loop
This is a problem if crossing over occurs within the inversion
A
B
C
A’
B’
C’
D
E
D’
E’
A-B-C-D-0-E’-C’-B’-A’
G-F-E-0-D’-F’-G’
F
G
‘F
G’
fragment
fragment
46
Inversions
•Paracentric inversion crosses
over with a normal chromosome,
the resulting chromosomes are an
acentric, with no centromeres,
and a dicentric, with 2
centromeres.
•The acentric chromosome isn't
attached to the spindle, so it gets
lost during cell division, and the
dicentric is usually pulled apart
(broken) by the spindle pulling the
two centromeres in opposite
directions. These conditions are
lethal.
•Pericentric inversion crosses
over with a normal chromosome,
the resulting chromosomes are
duplicated for some genes and
deleted for other genes. (They do
have 1 centromere apiece though).
•The gametes resulting from
these do not produce viable
progeny.
•
•Thus, either kind of inversion
has lethal results when it crosses
over with a normal chromosome.
•The only offspring that survive
are those that didn't have a
crossover or crossed over in
regions outside the inversion.
•Thus when you count the
offspring you only see the noncrossovers, so it appears that
47
crossing over has been
suppressed.
What are the consequences of crossing-over in an individual
homozygous for an inversion?
Genotype for normal individual
A
B
0
C
D
E
F
G
A
B
0
C
D
E
F
G
Genotype of an individual heterozygous for an inversion:
A
B
0
C
D
E
F
G
A
B
0
C
F
E
D
G
Genotype of an individual homozygous for an inversion:
A
B
0
C
F
E
D
G
A
B
0
C
F
E
D
G
48
Translocations
A segment from one chromosome is exchanged with a segment
from another chromosome.
Chromosome 1
A
B
C
D
E
F
----------------------0--------------------------------------------0----------------------A
B
C
D
E
F
Chromosome 2
O
P
Q
R
S
T
----------------------0--------------------------------------------0----------------------O
P
Q
R
S
T
Reciprocal translocation
A
B
C
D
S
T
----------------------0----------------------O
P
Q
R
E
F
----------------------0-----------------------
This is more specifically called a reciprocal translocation and
like inversions (and unlike duplications and deficiencies) no
genetic material is gained or lost in a reciprocal translocation.
49
Non-reciprocal translocations may also occur
long arms of chromosome 7 and
21 have broken off and
switched places. So you can
see a normal 7 and 21, and a
translocated 7 and 21.
This individual has all the
material needed, just switched
around (translocated), so they
should have no health
problems. However there can
be a problem when this person
has children.
Remember that when the
gametes are made, each parent
gives one of each chromosome
pair. What would happen if this
person gave the normal seven
and the 21p with 7q attached?
There are three copies of 7q
instead of two. And there is
only one copy of 21q
t(11;18)(q21;q21) translocation between chromosomes 11 and 18
at bands q21 and q21
Philadelphia chromosome: t(9;22)(q34;q11).
50
Pairing after translocations
As with inversions, individuals heterozygous for a reciprocal
translocation will exhibit abnormalities in chromosome pairing
A
B
C
D
E
F
----------------------0--------------------------------------------0----------------------A
B
C
D
S
T
O
P
Q
R
S
T
----------------------0--------------------------------------------0----------------------O
P
Q
R
E
F
Notice this individual has the normal amount of genetic material
(two copies of each gene).
However it is rearranged.
If the translocated fragment contains a centromere, you could get
dicentri and acentric chromosomes
How will translocated chromosomes pair in meiosis?
51
N1
F
F
E
E
T1
A
B
C
D
R
Q
P
O
A
B
C
D
R
Q
P
O
T2
S
S
T
T
N2
Homologous regions associate with one another.
These chromosomes will follow Mendel's rule of independent of
assortment. In this instance one must focus on the centromere
There are three possible patterns of segregation.
Normal Pairing of 10
chromosomes in maize
52
Chr8-9 translocation
Segregation after translocations
Alternate segregation:
ｷ
N1 and N2 segregate to one pole
ｷ
T1 and T2 segregate the other pole
These gametes have the normal haploid gene content: one copy
of each gene and are normal
Adjacent segregation:
ｷ
N1 and T1 segregate to one pole
ｷ
T2 and N2 segregate to the other pole
These gametes are anueploid: they are missing some genes and
duplicated for other genes.
Adjacent segregation
ｷ
N1 and T2 segregate to one pole
ｷ
N2 and T1 segregate to one pole
Therefore, in a translocation heterozygote, some of the
gametes are viable and some are inviable.
N1
F
F
E
E
T
1
A
B
C
D
R
Q
P
O
A
B
C
D
R
Q
P
O
T
2
S
S
T
T
53
N2
Reciprocal translocations result in genes that are known to map
to different chromosomes but behave as linked genes.
Under normal circumstances genes E and R assort independently
because they are on different chromosomes. However in a
translocation they will behave as closely linked genes and
segregate together.
N1
F
F
E
E
T1
A
B
C
D
R
Q
P
O
A
B
C
D
R
Q
P
O
T2
S
S
T
T
N2
54
Translocations (and inversion) breakpoints sometimes disrupt an
essential gene. That is the break occurs in the middle of a
gene.
In fact because of this, a number of specific translocations are
causally associated with specific human cancers.
The inherited disease Duchenne muscular dystrophy was mapped
through a translocation that specifically disrupted this gene.
55
abl/bcr
Fusion protein
Chronic myelogenous and acute lymphotic leukem
ALK/NPM Fusion
Large cell lymphomas
HER2/neu Fusion
Breast and cervical carcinomas
MYH11/CBFB Fusion
Acute myeloid leukemia
ML/RAR
Acute premyelocytic leukemia
Fusion
ERG/TMPRSS2
Fusion
prostate cancer
Gene fusion -prostate cancer -ERG merges with a prostatespecific gene called TMPRSS2. ERG is a transcription factors
56
3D chromosome/nucleus affects gross chromosomal alterations
Deletions/inv
ersions
Translocations
57
Xxxx
58