Genetics-Epigenetics Pg 21

Front 1

Genetic mechanisms can be categorized into
two classes.

Back 1

trans effects
cis effects

Front 2

trans effects

Back 2

include the loss or dysfunction of chromatin-associated factors which can in turn
alter chromatin structure and gene expression at certain genomic regions.

Front 3

cis effects

Back 3

represent mutations in noncoding regions that may be necessary for regulation. These
mutations, which may include the expansion of DNA repeats, can lead to chromatin alterations
that affect genome stability and gene expression

Front 4

uniparental disamony

Back 4

see pg 23

Front 5

angelman syndrome
prader willi syndrome

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see pg 24

Front 6

Epigenetic alterations involving DNA methylation can lead to cancer by various mechanisms

Back 6

Loss of DNA cytosine methylation (hypo) results in genome instability.
Focal hypermethylation
in gene promoters (hyper) causes heritable silencing and therefore inactivation of tumor
suppressor genes.
methylated CpG sites are hotspots for C→T transition mutations
caused by spontaneous hydrolytic deamination. Methylation of CpG sites also increases the
binding of some chemical carcinogens to DNA and increases the rate of UV-induced mutations

Front 7

Inactivation of tumor suppresor genes

Back 7

The first step of gene inactivation is shown as a localized mutation or gene
silencing by DNA methylation .The second hit is shown as either a loss of
heterozygosity (LOH) or transcriptional silencing by additional epigenetic events. In this
way, DNA methylation can contribute as one of the pathways to satisfy Knudson’s
hypothesis

Front 8

Tumor progression colon cancer

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Front 9

The earliest steps in tumorigenesis are depicted as

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abnormal clonal expansion, which
evolves during the stress of cell renewal. This is caused by factors such as aging and
chronic injury, from, e.g., inflammation. These cell clones are those at risk of subsequent
genetic and epigenetic events that would drive tumor progression.

Front 10

Abnormal epigenetic
events, such as aberrant gene silencing, could be the earliest heritable causes, in many
instances, for inducing

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the abnormal clonal expansion from within stem/progenitor cell
compartments in a renewing adult cell system

Front 11

gene silencing is triggered by
chromatin modifications that repress transcription, and the DNA hypermethylation of this
chromatin serves as

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the tight lock to stabilize the heritable silencing

Front 12

The major approaches currently underpinning ongoing cancer epigenetic
clinical trials

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consist of either DNA methyltransferase inhibitors to block
DNA hypermethylation or HDAC inhibitors to restore the acetylation status of key
histone amino acid residues

Front 13

Some of the most promising anticancer therapies include

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combinatorial use of DNMT1 and HDAC inhibitors

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Nucleoside analog inhibitors of DNA methylation

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The nucleoside analog inhibitors are incorporated into the growing cancer cell so that the DNA pattern of methylation is altered so perhaps tumor suppresor genes can be expressed again

Front 15

Bleeding disorders are a heterogeneous class of conditions in which blood
clotting is defective. They include both

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inherited and acquired disorders

Front 16

Fibrin is produced by action of

Back 16

the enzyme thrombin on the substrate
fibrinogen

Front 17

fibrin molecules are then cross-linked by action of the enzyme

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factor XIII

Front 18

Thrombin is activated by another clotting factor, factor

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Xa, which
acts on prothrombin

Front 19

Factor Xa is activated from factor X by either of two
pathways, referred to as

Back 19

extrinsic and intrinsic

Front 20

The extrinsic pathway involves

Back 20

tissue factor and factor VII, whereas the intrinsic pathway begins with factor
XII.!

Front 21

Clotting disorders can result from defects in platelet function or from

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defects in
the cascade of events that leads to fibrin formation

Front 22

Platelet disorders are more
likely to produce

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small punctuate hemorrhages or larger bruises on the skin and
mucous membranes

Front 23

Coagulation factor deficiency disorders typically result in

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bleeding episodes into joints and soft tissues.!

Front 24

Having less than 1% factor IX activity is consistent with a diagnosis of

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Hemophulia B

Front 25

Studies of platelet function include inspection of

Back 25

the blood smear for
abnormalities of platelet number or structure and measurement of bleeding
time, that is the time to formation of a platelet plug following vascular injury

Front 26

The Prothrombin time measures the activity of the extrinsic pathway from Factor VII through
production of fibrin, whereas the a Prothrombon Time measures

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the activity of the intrinsic
pathway through fibrin production.

Front 27

Finding an abnormality in aPTT but not PT
or platelet function implicates

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Factors XII, XI, IX, or VIII

Front 28

Some people have
inhibitors to clotting factors (e.g., antibodies), which can be determined by

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mixing a patient’s plasma and normal plasma If the patient has a clotting
factor deficiency, the clotting will be restored to normal in the mixture.

Front 29

If there
is an inhibitor in the patient's plasma, however, the inhibitor will continue

Back 29

to
interfere with clotting in the mixture. Finally, the specific deficiency can be
determined using assays for the individual factors.

Front 30

Hemophilia A (Factor VIII deficiency) and hemophilia B (Factor IX
deficiency) are clinically indistinguishable disorders characterized by

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prolonged bleeding after injury, tooth extractions, or surgery; bleeding into
joints or soft tissues, and frequently renewed bleeding after bleeding has
stopped

Front 31

Severe hemophilia is usually recognized in the first year of life,
frequently upon

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circumcision in males, but patients with milder disease may
not be diagnosed until they are more than 5 years old.

Front 32

Hemophilia B can be treated by

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IV infusion of recombinant or plasma derived clotting factors

Front 33

The degree of severity of hemophilia is related to the degree of factor deficiency. Severe
disease, in which spontaneous bleeding occurs, is associated with

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less than 1% of normal
activity

Front 34

Bleeding can also occur at mucous membranes, including

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gastrointestinal and
genitourinary tracts.!

Front 35

Hemophilia is most commonly treated by intravenous infusion of clotting factors either
prophylactically that is

Back 35

if bleeding is expected, as for a surgical procedure, or in response to
bleeding episodes

Front 36

The use of plasma-derived coagulation factors has provided therapy where there
previously was none, but it has come with its own problems, namely an extremely high
incidence of

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blood borne diseases, Hepatitis B, hepatitis C and HIV all occur in more than
half of patients with hemophilia

Front 37

The use of recombinant DNA-produced protein products
has eliminated the risk of

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transmission of human and animal infectious agents, but the
expense of protein replacement therapy (currently $50,000 to $100,000 per year) is
considerable

Front 38

individuals with hemophilia A and
with hemophilia B develop neutralizing

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antibodies (referred to as “inhibitors”) to the
replaced protein, reducing the therapeutic effect

Front 39

Factor IX deficiency and Factor VIII deficiency are

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X-linked disorders

Front 40

More than
2,100 different mutations have been described that result in Factor IX deficiency. The
majority are

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point mutations that lead to amino acid substitutions or the introduction of a
stop codon.

Front 41

It is not necessary to achieve normal levels of Factor IX
activity to achieve a therapeutic effect, as evidenced by

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carrier females with
low Factor IX levels but few or no symptoms

Front 42

Although the protein is normally produced
in the liver, other tissues, such as

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readily accessible muscle, may be able to act
as a factory for the production of Factor IX

Front 43

Gene therapy vectors evaluated for
hemophilia B have included

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adeno-associated virus (AAV), retrovirus,
adenovirus. AAV offers long-term stable expression

Front 44

One
limitation of AAV is that

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current vectors can only accommodate small genes,
however, the small size of the Factor IX coding sequence permits use of this
vector.!
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Front 45

AAV viral gene structured

Back 45

is a single-stranded DNA containing virus that does not cause human
disease. The viral genome consists of two genes, rep and cap, flanked by long
terminal repeats, which includes a promoter sequence

Front 46

The rep gene encodes
proteins required for replication of the virus and its integration into

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the
genome

Front 47

The cap gene encodes structural proteins for

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the viral capsid

Front 48

Both of
these genes can be removed and replaced with

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a gene of interest when the virus
is used as a gene therapy vector

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AAV
ordinarily integrates into a specific site on chromosome 19, but when used as a
gene therapy vector it may integrate at

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random into the host chromosome or
exist outside the chromosome as an episome

Front 50

Although gene therapy for hemophilia offers the promise of long-term therapy,
there are several potential risks that patients must carefully consider prior to
enrolling in a clinical trial. Risks include

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the possibility of germ line
transmission or insertional mutagenesis leading to the development of cancer. This involves integration of the virus adjacent to an oncogene, which would
activate the oncogene

Front 51

some results have suggested the production of functional Factor IX
protein following gene transfer, however, therapeutic levels of Factor IX have
not been reliably achieved. This may be in part due to

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the size of humans, as
compared to the preclinical animal models, making therapeutic doses difficult
to achieve

Front 52

Expression of the vector may be targeted
to the liver by incorporation of

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liver-specific promoter sequences adjacent to
the Factor IX gene and injection of the virus into the portal vein

Front 53

Plasmids containing expression cassettes can be introduced into the cell via

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naked DNA but efficiency of uptake is low, to improve uptake, plasmids are coupled with positively charged lipids

Front 54

Naked DNA, Cationic complexes, Gene Gun advantage

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lack of viral toxicity

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Naked DNA Cationic complexes disadvantage

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low transfection efficiency

Front 56

Cationic complexes characteristics

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DNA complex with lipids or polylysine

Front 57

Retroviruses contain

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single-stranded RNA that is copied into doublestranded
DNA in the cell. This DNA can enter the nucleus during cell division
and integrate at random sites into the host genome

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Retroviruses advantage

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stable, long-term expression in a dividing cell population

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Retroviruses danger

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inserted DNA may disrupt a host gene, including activation
of an oncogene if the inserted gene integrates nearby. This has occurred in
some children treated for X-linked severe combined immune deficiency

Front 60

Adenoviral vectors contain a double-stranded DNA which can infect

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dividing and non-dividing cells

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Adenoviral vectors disadvantage

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inserted DNA is not integrated into the
genome so the therapeutic benefit is not permanent, but insertional mutagenesis
does not occur

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Adenoviral vectors have been used in clinical trials, but there
have been problems due

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to toxicity and immune response to viral proteins

Front 63

Adeno-associated virus advantage

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It offers advantages of longterm
expression and low toxicity, but its use is limited by the relatively small
size (up to 4kb) of DNA that can be inserted

Front 64

Herpes vectors can
introduce large genes or even

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multiple genes as double-stranded DNA

Front 65

Lentiviral vectors are derived from the HIV
retrovirus and can infect both

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dividing and non-dividing cells

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Transfection of vectors into
cells that results in the production of specific short interfering RNA (siRNA) is
being explored for a variety of purposes, including

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silencing of genes involved
in viral fiction, cancer, and genetic disorders

Front 67

Gene regulation is not limited to control at the level of gene transcription.
There is another level of control that occurs post-transcriptionally, referred to
as

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RNA interference (RNAi).

Front 68

In experimental systems, and perhaps in some viral infections, RNAi begins
with introduction of double-stranded RNA molecules (dsRNA), which are
cleaved by the enzyme

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Dicer into siRNA molecules

Front 69

siRNAs separate into single strands and associate with specific proteins to form
the

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RNA-induced silencing complex (RISC). The single-stranded siRNA binds
to homologous sequences in mRNA and the RISC cleaves that RNA, thereby
in activating it.!
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