Genetics and Genomics in Medicine Chapter 9

Questions & Answers

Multiple Choice Questions

Question 9.1
Which, if any, of the following can be classified as a type of augmentation therapy?
a) Treatment using a small molecule drug to bind a target protein and prevent it working.
b) A bone marrow transplant.
c) Corrective surgery for cleft lip and palate.
d) Insulin treatment in diabetes.

Answer 9.1
b) A bone marrow transplant.
d) Insulin treatment in diabetes.

Question 9.2
With regard to treatment of inborn errors of metabolism (IEM), which of the following
statements, if any, is false?
a) IEMs are all single gene disorders that have been studied for many decades, leading to
the development of successful treatment in all cases.
b) IEMs can be treated by augmentation therapy, treatment to inhibit positively harmful
effects, or by prevention therapy.
c) Treatment for some individual IEMs can involve both augmentation therapy plus
treatment to inhibit positively harmful effects.
d) Treatment of some IEMs involves artificially forcing an increase in a minor metabolic
pathway to counteract a build-up in a toxic metabolite produced by a metabolic block in a
major metabolic pathway.

Answer 9.2
a) IEMs are all single gene disorders that have been studied for many decades, leading to
the development of successful treatment in all cases.

Explanation 9.2
For some IEMs there remains no suitable treatment.
Question 9.3
Concerning the efficacy of small molecule drugs, which, if any, of the following statements is
true?
a) At the level of clinical trials drugs can vary widely in how effective they are.
b) Once a drug has received regulatory approval, we can be sure that it will be effective in
all patients, although some people will receive more benefit from it than others.
c) Drugs used to treat psychiatric disorders are particularly effective.
d) Stains and beta blockers that were meant to reduce the risk of heart disease are good
examples of drugs that are largely ineffective.

Answer 9.3
a) At the level of clinical trials drugs can vary widely in how effective they are.

Question 9.4
Which of the following descriptions, if any, is false? A person’s ability to absorb or metabolize a
drug that is intended to treat a genetic disorder
a) is entirely due to genetic factors.
b) depends on a person’s lifestyle.
c) is not modified by having a bacterial infection.
d) is independent of a person’s diet.

Answer 9.4
a) is entirely due to genetic factors.
c) is not modified by having a bacterial infection.
d) is independent of a person’s diet.

Question 9.5
With regard to drug metabolism, which, if any, of the following statements, is true?
a) The therapeutic window is simply the range of plasma drug concentrations in which the
drug has therapeutic benefit.
b) Each individual drug molecule is metabolized by a specific drug-metabolizing enzyme
that is dedicated to the metabolism of that drug.
c) An ultrafast metabolizer is a person who metabolizes a drug too quickly and so is at risk
of an overdose
d) A poor metabolizer is a person who cannot metabolize a drug properly and is at risk of an
underdose.

Answer 9.5
None. All are false.

2
Explanation 9.5
a) The therapeutic window is the range of plasma drug concentrations in which the drug has
therapeutic benefit without causing extra safety risks due to drug toxicity.
b) Individual drug molecules can sometimes be metabolized by one of several drug-
metabolizing enzymes, and many drug-metabolizing enzymes metabolize multiple
different drugs.
c) An ultrafast metabolizer is a person who metabolizes a drug too quickly and so is at risk
of an underdose.
d) A poor metabolizer is a person who cannot metabolize a drug properly and is at risk of an
overdose.

Question 9.6
The diagram below shows the urinary metabolic ratio as a measure of CYP2D6 enzyme activity
in a total of about 700 individuals. After individuals were given a standard dose of a drug known
to be metabolized by CYP2D6 the metabolic ratio was obtained by measuring the urinary
concentration of the substrate drug and dividing it by the concentration of the metabolic product
resulting from CYP2D6 acting on the drug. Classify individuals with metabolic ratios in the four
ranges shown as (a) to (d) in terms of their drug-metabolizing abilities and describe the expected
genotypes associated with each group.

Metabolic ratio
(a) (b) (c) (d)

Answer 9.6
a) This falls within the range of ultrafast metabolizers who will have multiple CYP2D6
genes.
b) Extensive metabolizers with one or two normal CYP2D6 alleles.
c) Intermediate metabolizers two mutated CYP2D6 alleles, at least one of which makes
some gene product but at a reduced level.

3
 d) Poor metabolizers that have two null CYP2D6 alleles (homozygous deletion,
heterozygous deletion plus inactivating mutation, or inactivating mutations in both
alleles)

Question 9.7
Which of the following statements, if any, is false?
a) Monoclonal antibodies are made by identical immune cells and so will recognize and
bind just one specific epitope on a target molecule.
b) Monoclonal antibodies of rodent origin are far from ideal therapeutic agents because of
their short half-life in human serum and the potential for immune responses by the
recipient.
c) Humanized antibodies are hybrid antibodies that have constant regions of human origin
but variable regions of rodent origin.
d) An intrabody is an artificial constructs with just a single chain that is linked to variable
domains and, unlike regular antibodies with four polypeptide chains, has the potential to
work inside cells.

Answer 9.7
c) Humanized antibodies are hybrid antibodies that have constant regions of human origin
but variable regions of rodent origin.

Explanation 9.7
The variable domains in humanized antibodies are of human origin, except for the
complementarity-determining regions, which are of rodent origin.

Question 9.8
Which of the following statements, if any, is false?
a) Gene therapy involves the direct genetic modification of the cells of a person (or animal
model) to achieve a therapeutic goal.
b) Current gene therapy is directed at modifying somatic cells.
c) The only successful gene therapies are those in which cells are removed from a patient,
genetically modified, and then returned to the patient.
d) Gene therapy successes have largely involved treatment of recessively inherited
disorders.

Answer 9.8
c) The only successful gene therapies are those in which cells are removed from a patient,
genetically modified, and then returned to the patient.

4
Explanation 9.8
There have been some successes with in vivo gene therapy as well as ex vivo gene therapy.

Question 9.9
Concerning stem cells, which of the following statements is incorrect?
a) Stem cells occur frequently in our bodies.
b) A stem cell can divide asymmetrically to give a daughter stem cell plus a daughter transit
amplifying cell that can undergo a series of differentiation steps to give rise to a
differentiated cell.
c) If for any reason, stem cells are depleted, a stem cell can divide symmetrically to
regenerate the stem cell population.
d) In an adult person, stem cells are normally multipotent or unipotent.

Answer 9.9
a) Stem cells occur frequently in our bodies.

Question 9.10
Concerning transport of genes into human (or animal) cells, which, if any, of the following
statements is false?
a) Transduction means using viruses to transfer DNA into human (or other animal) cells.
b) Tropism refers to the ability of certain viruses to transduce only certain types of cell, such
as hepatocytes, but not neurons.
c) Tropism depends on a virus being able to recognize a specific receptor molecule on the
surface of the cell.
d) Transfection means transferring DNA into the cells by any means other than using
viruses.

Answer 9.10
None.

Explanation 9.10
All are correct.

Question 9.11
Concerning gene transfer into human cells, which, if any, of the following statements is false?
a) Integrating viruses can insert genes into the chromosomes of a host cell.
b) Most integrating viruses insert their DNA into a specific location within the genome.

5
 c) The great value of integrating viruses is that they allow foreign (and therefore,
therapeutic) DNA to be stably inherited so that it passes to all descendant cells of the
transduced cell.
d) Because non-integrating viruses cannot insert their DNA into the chromosomes of a cell,
the transduced DNA is quickly destroyed by enzymes within the host cell.

Answer 9.11
b) Most integrating viruses insert their DNA into a specific location within the genome.
d) Because non-integrating viruses cannot insert their DNA into the chromosomes of a cell,
the transduced DNA is quickly destroyed by enzymes within the host cell.

Question 9.12
Concerning animal models of human disease, which, if any, of the following statements is false?
a) Primates should be the best animal models, but for mostly practical reasons, rodent
models have been preferred.
b) Rats have been the preferred disease models because they offer the best balance between
rapid breeding, size and the cost of maintaining colonies.
c) Rodent models are especially suited to modelling neuropsychiatric disorders.
d) All animal models have limitations regarding how far we can make inferences to help
understand human disease.

Answer 9.12
b) Rats have been the preferred disease models because they offer the best balance between
rapid breeding, size and the cost of maintaining colonies.
c) Rodent models are especially suited to modelling neuropsychiatric disorders.

Explanation 9.12
Mice have been the preferred disease models, but rodent models are poorly suited to modelling
neuropsychiatric disorders (in part, because they are not good models of cognitive capacity).

Question 9.13
Concerning making animal models of human disease, which, if any, of the following statements
is false?
a) Pronuclear microinjection is a general way of making a transgenic animal and involves
microinjection of foreign DNA into a fertilized egg cell.
b) Pronuclear microinjection is best suited to modelling recessively inherited single gene
disorders.
c) Gene targeting using embryonic stem cells depends on having well-characterized
embryonic stem cell lines that can readily allow transmission through the germ line.

6
 d) Gene targeting using embryonic stem cells in mice is a popular way of modelling disease
phenotypes that result from a gain-of-function.

Answer 9.13
b) Pronuclear microinjection is best suited to modelling recessively inherited single gene
disorders.
d) Gene targeting using embryonic stem cells in mice is a popular way of modelling disease
phenotypes that result from a gain-of-function.

Explanation 9.13
Pronuclear microinjection is not suited to modelling recessively inherited single gene disorders,
but has often been used to model disease phenotypes that result from a gain-of-function. Gene
targeting using embryonic stem cells in mice is not well suited to modelling disease phenotypes
that result from a gain-of-function but is a popular way of modelling the loss-of-function in
recessively inherited single gene disorders.

Question 9.14
Which, if any, of the following statements is false?
a) Hematopoietic stem cells are multipotent because they can give rise to a variety of
different cell types.
b) Mammalian embryonic stem cell lines are pluripotent because they can give rise to all
types of cell in the body.
c) Transdifferentiation is a type of epigenetic reprogramming in which a differentiated cell
is induced to become pluripotent.
d) A transit amplifying cell is a cell produced by asymmetric division of a stem cell and has
the potential to give rise to differentiated cells.

Answer 9.14
c) Transdifferentiation is a type of epigenetic reprogramming in which a differentiated cell
is induced to become pluripotent.

Question 9.15
Which, if any, of the following statements is false?
a) Transdifferentiation means reprogramming of a differentiated cell so that it acquires the
characteristics of another type of differentiated cell.
b) In dedifferentiation a differentiated cell is artificially reprogrammed so that it behaves as
a pluripotent cell.
c) Human induced pluripotent stem (iPS) cell lines are usually generated by artificial
dedifferentiation of readily accessible human cells, such as skin cells.

7
 d) Human iPS cell technologies do not offer clinical applications but they are of value for
studying pathways of cellular differentiation

Answer 9.15
d) Human iPS cell technologies do not offer clinical applications but they are of value for
studying pathways of cellular differentiation

Explanation 9.15
Human iPS cell technologies have the potential for valuable clinical applications, most readily in
creating cellular disease models and possibly in extending ex vivo gene therapy.

Question 9.16
Which, if any, of the following statements is true?
a) Autologous cell transplantation is involved in in vivo gene therapies: cells from an
individual are genetically modified and then returned to that individual.
b) Adenovirus vectors have the advantage that they offer very high level expression and are
well suited to gene therapy for blood disorders.
c) Adenovirus vectors have a better safety profile than adeno-associated virus vectors and
have a larger insert size capacity.
d) Adeno-associated virus vectors are well suited to gene therapy for blood disorders but
have a low insert size capacity

Answer 9.16
None.

Explanation 9.16
All are false.
Autologous cell transplantation is involved in ex vivo gene therapy. Both adenovirus vectors and
adeno-associated virus vectors are not well suited to gene therapy for blood disorders because
neither of them are integrating vectors (because blood cells have short lives some kind of
retroviral integrating vectors are needed for gene therapy for blood disorders – the hope is to
insert the gene constructs into the chromosomal DNA of hematopoietic stem cells). Adenovirus
vectors have a poor safety record because they often induce powerful inflammatory responses in
the recipient of therapy.

Question 9.17
Which, if any, of the following statements is false?
a) The great majority of clinical gene therapy trials have had limited success
b) The only successful gene therapies have been for recessive blood disorders.

8
 c) The only successful gene therapies have been ex vivo gene therapies.
d) Gene therapy for inherited disorders represents a minority of clinical gene therapy trials.

Answer 9.17
b) The only successful gene therapies have been for recessive blood disorders.
c) The only successful gene therapies have been ex vivo gene therapies.

Explanation 9.17
There have been examples of other successful gene therapies that involve brain disorders and in
vivo gene therapy for eye disorders, for example.

Question 9.18
Which, if any, of the following statements is false?
a) RNA interference (RNAi) is a cellular defense mechanism that is triggered by the
presence in cells of unnatural double-stranded RNA, as can occur after viral infections.
b) RNAi therapy is a type of RNA-targeted therapy in which specific double-stranded RNA
constructs are engineered to appear in diseased cells in order to incite the cells to destroy
any RNA that contains the same sequence.
c) By destroying RNAs that are related to a specifically introduced genetic construct,
artificial RNAi is effectively a type of gene-specific silencing.
d) RNAi therapy is best suited to silencing genes so as to replicate a phenotype caused by
loss-of-function mutations.

Answer 9.18
d) RNAi therapy is best suited to silencing genes so as to replicate a phenotype caused by
loss-of-function mutations.

Explanation 9.18
RNAi is a convenient way of silencing a gene in cultured cells as a way of understanding its
function but RNAi therapy is better suited to specific silencing of a positively harmful gene in
diseased cells than it is to replicating loss-of-function phenotypes.

Question 9.19
Which, if any, of the following statements is false?
a) Genome editing means making a predetermined change to the nucleotide sequence at just
one locus within an intact cell.
b) The specificity of genome editing depends on an initial site-specific cleavage of double-
stranded DNA following base pairing with specifically designed nucleotide sequences.

9
 c) Genome editing has the potential to permit specific “gene correction” in which a mutant
sequence in a cell is restored to the normal sequence.
d) Genome editing might also have therapeutic potential by specifically inactivating a gene
in some cases.

Answer 9.19
b) The specificity of genome editing depends on an initial site-specific cleavage of double-
stranded DNA following base pairing with specifically designed nucleotide sequences.

Explanation 9.19
The site-specific cleavage is also often carried out following recognition of the sequence at the
target locus by a combination of zinc fingers within zinc finger nuclease proteins.
d) See the example of therapeutic genome editing by zinc finger nucleases to inactivate the
CCR5 gene as a cure for HIV-AIDS (see Box 9.3 on page 365.)

Question 9.20
Which, if any, of the following descriptions is false?
a) Zinc fingers are elements of protein secondary structure in which the polypeptide chain
folds back upon itself after co-ordination of a Zn2+ ion with selected amino acids, often a
pair of cysteines and a pair of histidines.
b) Zinc finger nucleases are natural proteins containing a sequence of zinc fingers that can
bind to specific sequences in DNA.
c) After zinc finger nucleases bind to both DNA strands at a specific DNA sequence they
attract cellular DNA cleavage enzymes, inducing them to make a double-stranded break
at just that one position in the genome.
d) The CRISPR-Cas system also allows genome editing but in this case the target DNA
sequences are recognized by guide RNA sequences rather than proteins.

Answer 9.20
b) Zinc finger nucleases are natural proteins containing a sequence of zinc fingers that can
bind to specific sequences in DNA.
c) After zinc finger nucleases bind to both DNA strands at a specific DNA sequence they
attract cellular DNA cleavage enzymes, inducing them to make a double-stranded break
at just that one position in the genome.

Explanation 9.20
Zinc finger nucleases are not natural: they are proteins produced after genetic engineering to
covalently join DNA sequences that can specify a series of zinc finger modules to a bacterial
DNA sequence that can specify a DNA cleavage domain.

10
Fill in the Blanks Questions

Question 9.21
Fill in the blanks with single words.

In some diseases the problem is the loss of some function and a type of ____1_____ therapy is
used to supplement the resulting deficiency. It may be a deficiency in some normal aspect of the
____2_____, such as deafness, a deficiency of organs or ____3____ , or a deficiency of
molecules (which might be at the level of ____4_____ , ____5_____ , or downstream factors).
Sometimes, however, disease is not due to a deficiency; instead, the problem is that there is some
positively _____6_____ effect produced at some level (at the level of the phenotype,
____3_____ or ____4______ ), that cannot be supplemented. Here treatment is possible by
seeking to eliminate, correct or ____ 7_____ the agent causing the positively _____6_____
effect. The treatment might seek to kill dangerous _____3____ , for example, or to ____7_____
a _____6_____ _____4_____ or a _____7_____ _____6_____. In the latter case, the treatment
might often be to use some kind of _____8_____ , such as a conventional ____9_____ molecule
____8_____ that usually works by binding to a cleft in the _____6_____ _____5_____ and
thereby ____7____ its ____6____ effect. A third class of disease treatment seeks to use a
____8____ in order to alter a person’s ______10_______ to the disease, or to alter exposure to
some _____11_____ factor.

Answer 9.21
1. augmentation. 2. phenotype. 3. cells. 4. genes. 5. protein(s). 6. harmful. 7. inhibit. 8. drug. 9.
small. 10. susceptibility. 11 environmental.

Question 9.22
Fill in the missing blanks with single words.

In the recent past, virus vectors used in gene therapy trials were often based on a type of
retrovirus called a ______1_______retrovirus. They had the advantage of allowing a
_____2_____ DNA to be stably inserted into the _____3____ DNA of cells. For cells that are
short-lived, such as blood cells, the hope was that a certain percentage of _____4_____ cells
might be successfully transduced so that there was a self-renewing population of cells carrying
the desired _____2_____ DNA. Unfortunately, vectors based on ______1_______retroviruses
have a poor safety profile: there is little control over where they ______5_______ into the
_____3____ DNA and sometimes when they _____5_____ they activate a neighboring
_____6_____, causing _____7_____. As a result, in modern gene therapy trials it is now

11
commonplace to use self-_____8______ strains of a class of retrovirus vectors known as
_____9____ that are much safer to use.

Answer 9.22
1. gamma. 2. therapeutic. 3. chromosomal (or genomic). 4. stem. 5. integrate. 6. oncogene. 7.
cancer. 8. inactivating. 9. lentiviruses.

Question 9.23
Fill in the missing blanks with single words.

Genome _____1______ means artificially introducing a specific change in the DNA sequence at
a unique, pre-determined location within the genome of an ____2____ cell. The method relies on
some form of recognition of specific sequences on both DNA strands at a locus that then allows
an artificially introduced _____3____-stranded DNA break at this location. In response to the
_____3____-stranded DNA break, DNA repair is carried out by the cell but after using
non_____4_____ end joining DNA repair, errors can be made in the repair that can occasionally
result in a desired specific DNA change. In one system genetically engineered ______5_____
______6_____ nucleases are used in which a DNA is constructed to code for a specific sequence
of ______5______ _____6______ and is then ligated to a DNA sequence that will specify a
DNA ______7______ domain. A plasmid containing the resulting DNA construct can encode a
______5______ _____6______ nuclease when transfected into a cell. Using this technology, a
pair of ______5______ _____6______ nucleases can be designed to bind to specific sequences
on the opposite DNA strands at a desired unique position in the genome and the adjoining DNA
cleavage domains work to produce the required ____3_____–strand DNA break.

Answer 9.23
1. editing. 2 intact. 3. double. 4. homologous. 5. zinc. 6. finger. 7. cleavage.

12
Essay and Listing Questions

Question 9.24
There are three broad classes to disease treatment. Give a brief outline of the three classes.

Answer 9.24
1) Augmentation therapy. Designed to supplement a deficiency of some bodily function, or
a failing organ/loss of cells, or a defective or missing gene, or of some downstream
metabolite. Can involve providing some artificial aid to restore the bodily function (a
hearing aid, for example), or a supply or healthy organs/cells (transplantation), or a
supply of properly working genes (gene therapy), or gene products/downstream factors
(for example, insulin).
2) Treatment to eliminate, correct, or inhibit some positively harmful effect. That can
involve corrective surgery, killing of harmful cells (using antibiotics or by provoking an
increased immune response), inhibiting gene expression, or by blocking the function of a
harmful protein (such as by using conventional small molecule drugs or monoclonal
antibodies to specifically bind to the harmful protein, thereby disrupting its function).
3) Prevention therapy. The idea is to give some treatment that reduces the risk of developing
disease, such as giving statin drugs to reduce blood pressure and so lessen the
susceptibility of developing various cardiovascular diseases.

Question 9.25
Four stages are often identified in drug development: a preclinical stage plus three clinical trial
stages. What is involved in these?

Answer 9.25
• The pre-clinical stage involves a battery of laboratory tests that can be in both cultured
cells (testing the efficacy of the proposed treatment when there is a suitable cellular
assay) and in animal models where efficacy and toxicity are assessed as well as the
pharmacokinetic parameters (studies of the absorption, activation, catabolism, and
elimination of the drug).
• Phase I clinical trials involve small-scale studies of healthy volunteers (typically, up to
100 people) and are largely a study on the safety of the treatment. Both the
pharmacokinetics and also pharmacodynamics (the response of a target organ or cell to a
drug) are assessed.
• Phase II clinical trials represent the first clinical trials on patients. Usually several
hundred patients are treated; both safety and efficacy of the treatment are monitored.

13
 • Phase III clinical trials represent large-scale patient trials. Typically the effect of
treatment on thousands of patients in multiple centers are assessed in randomized,
controlled trials. Again, the objective is to assess both the safety and efficacy of the
treatment.

Question 9.26
Many of the genes that produce the enzymes and other proteins involved in handling drugs are
polymorphic. Why should that be?

Answer 9.26
From the body’s perspective, drug metabolism is really a defense mechanism: the body’s priority
is to facilitate excretion of the parent drug and its metabolites, and so limit their ability to
accumulate within the body and cause dose-dependent toxicity. We have a range of genes, many
of them polymorphic, that make the proteins that deal with xenobiotics, foreign chemical
substances that we ingest but that are not normally part of the human diet. The polymorphism
developed initially as a form of self-protection to reduce the risk from ingested toxins (such as
from certain plants and fungi). It was driven by natural selection because if a person has a wider
range of proteins that can interact with potentially harmful xenobiotics, there is a reduced chance
that the person is seriously affected by ingested harmful substances.

Question 9.27
What distinguishes phase I and phase II reactions in the metabolism of small molecule drugs?

Answer 9.27
Small molecule drugs are based on hydrocarbon backbones and so are lipophilic, but drug
metabolism allows them to be converted into hydrophilic forms that are easier to excrete from
the body.
Phase I reactions are usually carried out by monooxygenases; these work by adding an oxygen
atom from molecular oxygen to produce a more polar substance. Often a hydroxyl group is
introduced, or a bulky alkyl group bound to a nitrogen, sulfur, or oxygen atom is replaced by a
hydrogen atom. The drug derivative is typically left with a more reactive group, a molecular
‘handle’ that makes it easier for a secondary reaction to be carried out (see below).
Phase II reactions are conjugation reactions, catalyzed by transferases that add one of a variety of
chemical groups, notably acetyl, methyl, glucuronyl, glutathionyl and sulfate groups. Phase II
reactions commonly occur after phase I reactions have introduced a molecular handle for
attaching the secondary chemical group. A hydroxyl group attached during phase I, for example,
provides a convenient site for an acetyl group or a sugar (glucuronyl) group to be attached by a
phase II enzyme, detoxifying the drug and assisting in its excretion.

14
Question 9.28
Using the example of genes encoding cytochrome P450 enzymes, illustrate how genetic variation
in drug-metabolizing enzymes can often stem from gene copy number variation.

Answer 9.28
Probably, the best example of copy number variation for genes encoding drug-metabolizing
enzymes comes from the CYP2D6 locus on chromosome 22 where the number of gene copies
can range from 0 to 13. People who lack both CYP2D6 alleles or who have a deletion of one
CYP2D6 allele and an inactivating mutation in the other allele are poor metabolizers for the
drugs that this enzyme normally handles. Those who have a solitary CYP2D6 allele that has a
reduced function are intermediate metabolizers. Some individuals have many CYP2D6 genes and
are ultrafast metabolizers.

Question 9.29
In some cases, genetic variation at multiple loci is known to affect the response to a specific
drug. What is known about genetic variation that affects our responses to the anticoagulant
warfarin?

Answer 9.29
At least three loci are involved: CYP2C9, VKORC1 and CYP4F2, as listed below.
1) CYP2C9 makes a major cytochrome p450 enzyme that is known to be involved in
metabolizing multiple drugs. The CYP2C9 enzyme hydroxylates warfarin to produce 7-
hydroxywarfarin.
2) VKORC1 makes the C1 subunit of the vitamin K epoxide reductase complex (VKOR).
The latter enzyme works to ensure that there is a healthy supply of vitamin K, a vitamin
that is essential for activating four important blood clotting factors, Factors II, VII, IX
and X).
3) CYP4F2 makes an enzyme that works as a vitamin K oxidase that converts vitamin K
quinone to hydroxyvitamin K.

Question 9.30
Sometimes, a prescribed drug can be dangerous, and occasionally deadly, according to a
patient’s genotype. Give three examples of such.

Answer 9.30
1) Suxamethonium (succinylcholine) works as a fast-acting muscle relaxant and is used
before surgery. Normally the effects of the drug wear off quite quickly when the drug is
metabolized by the enzyme butylcholinesterase. Low metabolizers are at risk of apnea—

15
 they remain paralyzed and unable to breathe after surgery because they cannot regain
their muscle function quickly enough and may require extended ventilation.
2) 6-mercaptopurine and azathioprine are immunosuppressant drugs that are important in
dampening down potentially harmful immune responses after organ transplantation. The
enzyme thiopurine S-methyltransferase (TPMT) inactivates these immunosuppressant
drugs by adding a methyl group. In people with two low-activity TPMT alleles, the drugs
are metabolized slowly; if normal doses are given, the drugs accumulate and can result in
life-threatening bone marrow toxicity.
3) Various statins and the inhalation anesthetics halothane and isoflurane are associated with
usually mild myopathies but in some patients there can be severe muscle toxicity in
which the muscle tissue breaks down (rhabdomyolysis) and can lead to death. Persons
with inactivating mutations in the ryanodine receptor gene develop life-threatening
rhabdomyolysis and an extreme rise in temperature, a form of malignant hyperthermia
(OMIM 145600).

Question 9.31
The CFTR gene that underlies cystic fibrosis was isolated by positional cloning in 1989. Twenty
years later, Jack Riordan, one of the major contributors to this historic achievement, was quoted
as saying “the disease has contributed much more to science than science has contributed to the
disease”. What did he mean by this, and what important developments have occurred in treating
cystic fibrosis since 2009?

Answer 9.31
He meant that when the novel CFTR gene was first identified, almost nothing was known about
how it worked. The CFTR protein was subsequently shown to function as a channel that
regulates transmembrane conductance by allowing chloride ions to pass through the cell
membrane. A great deal was also discovered about aspects of CFTR biology that would inform
diverse fields such as protein trafficking and membrane transport.
In contrast to the burgeoning information on how the CFTR protein works, no successful
treatment had been devised by 2009. Gene therapy for cystic fibrosis has enormous problems in
terms of gene delivery and expression (because of the thick mucus layer coating the surface of
lung epithelia in cystic fibrosis patients). Another problem was that six different classes of
mutations could be identified to be associated with the disease, according to their effects on how
the gene normally works in cells. That meant that conventional small molecule drug treatment
needed to deal with different types of molecular pathogenesis.
Recently, there has been a little progress. In 2012 ivacaftor (marketed as Kalydeco by Vertex
Pharmaceuticals) became the first drug approved by the US Food and Drug Administration to
target a cause of cystic fibrosis rather than the condition’s symptoms. It has been targeted to treat
patients with the G551D mutation, which causes the chloride channel to fail to open. Ivacaftor
works by helping to reopen the chloride channel. While ivacaftor may well result in marked

16
improvement in longevity, quality of life, treatment burden, and so on, it is directed at just this
one mutation, and is applicable to just 4% of cystic fibrosis patients (those who have at least one
G551D mutation).

Question 9.32
There are two broad principles regarding the technological aim of somatic gene therapy: (a)
genetically modifying disease cells (without killing them), and (b) killing disease cells either
directly or indirectly. Explain what is involved in the two strategies.

Answer 9.32
1) The idea is to alter gene expression for therapeutic benefit. That usually means adding a
supplementary cloned gene copy to make some desired product (that is lacking in
diseased cells of the patient). In principle, it could also mean inactivating or inhibiting
how a mutant gene works for therapeutic benefit, or correcting a gene so that it is restored
to its normal function.
2) Killing disease cells is particularly appropriate in cancer gene therapies. It can be done
directly by transferring into cancer cells a recombinant DNA that can make some
cytotoxic agent, such as ricin, or by genetically modifying non-disease cells with the aim
of provoking a strong immune response against the disease cells.

Question 9.33
In mammals, pluripotent cells occur naturally in the early embryo but pluripotent cell lines can
also be artificially created. The first approach was to make embryonic stem cell lines from cells
isolated from early embryos. More recently, pluripotent cell lines have been made by artificially
changing the epigenetic settings of differentiated cells. What is involved in the latter case?

Answer 9.33
Artificial epigenetic reprogramming can mean transdifferentiation (when one type of
differentiated cell is induced to change into a different type of differentiated cell, such as
changing a skin fibroblast to a neuron, for example), or dedifferentiation (when a differentiated
cell is induced to change to a less specialized cell). The reprogramming can be carried out by
transferring genes encoding the relevant transcription factors needed to induce the desired
change, or by providing purified transcription factor proteins or, sometimes, specific chemicals
that can induce the production of the required transcription factors.

Question 9.34
Describe the characteristics of two viral vectors based on RNA genomes and two viral vectors
based on DNA genomes that are used in gene therapy.

17
Answer 9.34
• Gammaretroviruses. These are simple, single-stranded RNA viruses that have a reverse
transcriptase which allows them to make a cDNA copy that can integrate into
chromosomes of the host cell. They cannot pass though the intact nuclear membrane and
so can infect dividing cells only (when the nuclear membrane dissolves). Vectors based
on gammaretroviruses have a checkered safety record because they quite frequently
integrate close to proto-oncogenes causing leukemia.
• Lentiviruses. These are complex single-strand RNA viruses that are also able to make
cDNA copies that integrate into chromosomes but they can pass through the nuclear
membrane and so can infect both dividing and non-dividing cells. Vectors based on
lentiviruses have a good safety profile in gene therapy.
• Adenoviruses. They are complex double-stranded DNA viruses that can infect both
dividing and non-dividing cells but are non-integrating. Their big advantage is that they
offer high level expression but they are not stably inherited by the daughter cells of
dividing cells. Vectors based on adenoviruses have the big disadvantage that they can
elicit powerful inflammatory/immune response in patients.
• Adeno-associated viruses. They are comparatively simple, single-stranded DNA viruses.
They can infect both dividing and non-dividing cells. They offer high level expression
and many strains have useful tropism characteristics. Vectors based on adeno-associated
viruses have a much better safety record than adenoviruses.

Question 9.35
Hematopoietic stem cells can be important target cells in gene therapy for blood disorders and
some other disorders, including certain brain disorders. Explain the significance of hematopoietic
stem cells and how they are exploited in gene therapy.

Answer 9.35
Hematopoietic stem cells (HSC) form in the bone marrow and give rise to all classes of blood
cells, including macrophages, monocytes, granulocytes, platelets, erythrocytes, B lymphocytes
and T lymphocytes (where precursor cells migrate to the thymus). In addition, HSC give rise to
certain tissue immune system cells, such as microglia (the resident macrophages of the brain and
spinal cord) and dendritic cells (a class of immune system cells that work in presenting foreign
antigens to T cells and that are found in various types of tissue).
Being stem cells, HSC serve as an immortal source of the cells listed above and even a single
HSC is believed to be able to re-populate all blood cells. Although the greatest concentration of
HSC occurs in the bone marrow they are also found infrequently in peripheral blood. Ex vivo
gene therapy for recessive blood disorders typically involves using enrichment techniques to
obtain cells from the patient that are enriched in HSC. That can involve using monoclonal
antibodies specific for the CD34 antigen to select for cells containing this antigen (which is a

18
marker of HSC). The enriched cell population is then typically transduced with a recombinant
retroviral vector such as a lentivirus vector that contains a normal version of the gene that is
defective in patients.
Gene therapy has also been possible for certain brain disorders, such as X-linked
adrenoleukodystrophy, a lipid storage disorder that primarily affects the brain. That was possible
because the transduced HSCs were able to give rise to myelomonocytic cells (with
characteristics of both granulocytes and monocytes) that migrated into the central nervous
system to replace diseased microglial cells and relieve the lipid storage problem.

Question 9.36
What possible clinical applications might be derived from induced pluripotent stem cell
technologies?

Answer 9.36
From a medical perspective, induced pluripotent stem (iPS) cell technologies have two
potentially exciting applications as described below.
1) Provision of human cellular models of disease. Animal disease models have been very
valuable because they allow the use of invasive studies to understand the molecular basis
of human disease. But they are only models; they quite often show important differences
from humans. Accessible skin cells from a patient can now be reprogrammed to become
cells that can then be directed to differentiate into cells relevant to the disease process
(such as normally inaccessible neurons for a neurodegenerative disorder). The genetically
impaired disease cell lines will be useful for drug screening (testing for toxicity, efficacy,
and so on) and for studying the molecular basis of disease in human cells.
2) Therapeutic applications. Accessible cells from a patient can be artificially
dedifferentiated to make iPS cells that are then genetically modified, and then returned to
the patient without provoking an immune response. Successful environmentally induced
reprogramming of human cells may transform the prospects of using dedifferentiated
human cells therapeutically and there is the potential of directing the genetically modified
cells to form cells of a desired cell type.

Question 9.37
Molecular therapeutic strategies sometimes target RNA instead of DNA. What is involved in
RNA interference therapy, and how useful has it been?

Answer 9.37
RNA interference therapy is a type of artificial gene silencing. Different diseases are potentially
amenable to treatment based on gene silencing (in which the expression of the gene is artificially
repressed in some way). In some cases, the disease is due to a gain-of-function mutation or to a

19
dominant-negative effect: the problem is a mutant resident gene that is doing something
positively harmful. Here, the strategy must be to selectively inhibit the expression of the mutant
gene, without affecting the normal allele too much. (There are therefore parallels with treating
infectious diseases, which might be treated by targeting a pathogen-specific gene or gene
product).
RNA interference is a natural gene-silencing phenomenon that cells use as a protective
mechanism to counteract viruses and to limit the spread of transposons in the germ line.
Artificial RNAi-mediated gene silencing usually involves designing two short
oligoribonucleotides to have complementary sequences so that they form a siRNA (short
interfering RNA) duplex that is transfected into diseased cells. Alternatively, a gene is
transfected that can make a short hairpin RNA which is processed in transfected cells to give the
desired siRNA duplex. One of the sequences of the siRNA is chosen to be identical to, and
specific for, the target RNA that needs to be silenced. In the cell one of the two complementary
RNA sequences will bind to the target RNA. The unnatural double-stranded RNA prompts the
cell to degrade the RNA.
RNAi therapy is not straightforward: complete gene silencing is difficult to obtain. There can
also be the risk of off-target effects, in which very closely related sequences that occur by chance
in other genes become collateral targets. A variety of clinical gene trials have been or are being
carried out. Although the therapeutic potential of RNAi therapies might be high, the technology
needs to be refined.

Question 9.38
How has exon skipping therapy been applied to treat Duchenne muscular dystrophy?

Answer 9.38
Internal deletions are a common cause of pathogenesis in the dystrophin gene, and many
deletions that affect central exons result in a frameshift, resulting in severe Duchenne muscular
dystrophy (DMD). However, quite large deletions within the central region of the gene are
associated with mild Becker muscular dystrophy (BMD) if they do not change the translational
reading frame. These large in-frame deletions do not result in severe disease because the
sequence of a large central part of the dystrophin protein is unimportant (certainly when
compared to the N-terminal and C-terminal regions which are functionally vital components of
the protein and are unaffected by a large in-frame central deletion).
Exon skipping therapy seeks to restore the translational reading frame by inducing altered
splicing so that at the RNA level a central exon or exons is omitted from the long dystrophin
mRNA to produce an effect that is similar to the effect of a central in-frame deletion associated
with milder BMD. In total about 25% of DMD-associated deletions might have the translational
reading frame restored by inducing skipping of just one internal exon, exon 51, and that would
make treatment available to 15% of DMD patients. Skipping of other central exons can further
extend this type of treatment.

20
Local intramuscular injections of an antisense oligonucleotide to induce skipping of exon 51 has
been shown to restore dystrophin production in muscle fibers of patients with the appropriate
types of dystrophin exon deletion. Follow-up systemic administration of the oligonucleotide
(with access to the circulation via abdominal subcutaneous injections) did not elicit any serious
adverse reactions and the procedure seems to work quite well. In 10 of the 12 treated boys (aged
7 to 13 years), new dystrophin expression was observed in between about 60% and 100% of
muscle fibers, and clinical benefit was significant as measured by improved walking statistics
when compared with controls.
The oligonucleotides that are used in therapeutic exon skipping need to be more stable than
conventional oligonucleotides. Serepta Therapeutics uses phosphorodiamidate morpholino
oligonucleotides; Prosensa uses 2ʹ-O-methyl-modified ribose molecules with a full-length
phosphorothioate backbone. Even then, the oligonucleotides have a limited half- life after
systemic administration (for example, 29 days in the latter case).

Question 9.39
Genome editing is being used in an attempt to cure HIV-AIDS. What is the experimental strategy
towards achieving that goal?

Answer 9.39
The human immunodeficiency virus HIV launches its attack on the body by infecting CD4
helper T cells. By attacking and killing helper T cells (regulatory immune system cells with a
major role in helping to protect us against viruses), HIV compromises the immune system;
people with AIDS are unable to fight off common infections and they develop various virus-
induced cancers.
To latch onto a helper T cell, HIV first binds to a CD4 receptor on a T cell and then interacts
with a co-receptor—often the chemokine (C–C motif) receptor 5 (CCR5). Unlike CD4, the
CCR5 receptor is not so important in T-cell function, and some normal people (about 5–14% of
individuals of European descent) have defective CCR5 receptors because they have a CCR5
allele with an inactivating 32 bp deletion (CCR5-Δ32). Heterozygotes with one CCR5-Δ32 allele
are more resistant to HIV infection than the normal population, and homozygotes are highly
resistant to HIV infection.
The idea that HIV-AIDS could be cured by artificially inactivating CCR5 was promoted by the
famous ‘Berlin patient’ study. First reported in 2009, an HIV patient with acute myeloid
leukemia received allogeneic CD34+ peripheral blood stem cells from an HLA-identical donor
who had been screened for homozygosity for the CCR5-Δ32 allele. Four years later, and after
discontinuation of anti-retroviral therapy, the patient appears to be free from HIV, indicating that
this could be the first complete cure for HIV infection.
The Berlin patient study was clearly an exceptional situation, and various follow-up studies have
sought to extend resistance to HIV by inactivating CCR5 in autologous T cells. Among them are

21
phase I and phase II gene therapy trials carried out by Sangamo Biosciences in which genome
editing is applied using zinc finger nucleases to target and inactivate the CCR5 gene.

Question 9.40
How might a form of germ-line gene therapy be used to treat severe mitochondrial diseases?

Answer 9.40
Mutations in mitochondrial DNA (mtDNA) are a significant cause of human disease: pathogenic
mutations are found in at least 1 in 200 of the population, and cause severe multisystem disease
in approximately 1 in 10,000 of the population. Pathogenic mtDNA can be maternally inherited,
but there are no effective treatments for mitochondrial DNA disorders.
In the clinical management of mtDNA disorders, the emphasis has therefore been on prevention.
Preimplantation and prenatal diagnosis are well established in clinical genetic practice as a way
of selecting unaffected embryos. However, the results can be difficult to interpret for patients
with heteroplasmic mtDNA mutations (when there may be variable numbers of mutant and
normal mtDNAs in each cell). In addition, an increasingly large group of diseases are recognized
to be caused by homoplasmic mtDNA mutations (all the mtDNA molecules are mutant). Here,
prevention is not an option—all the offspring would inherit the pathogenic mutation in the
maternal egg, and this type of genetic defect can be associated with a very high disease
recurrence risk.
An entirely different way of trying to prevent the transmission of homoplasmic mutations is to
replace maternal mtDNAs by mtDNAs from an asymptomatic donor. This type of approach has
been used in mouse and primate models, with encouraging results. Two recent studies that used
slightly different approaches have been carried out in human embryos in vitro.
In the pronuclear transfer technique an oocyte with mutant mtDNA is fertilized; the normal
karyoplast (combined male and female pronuclei) is isolated and then transferred into an
enucleated donor zygote with normal mitochondria. In the metaphase II spindle transfer
technique the spindle is transferred from an oocyte that has mutant mtDNA into a mitochondrial
donor oocyte followed by intracytoplasmic sperm injection fertilization. Both techniques involve
monitoring embryo development following transfer. The resulting human embryos appear to be
viable in vitro, and the degree of mutant DNA carryover is low or undetectable.
The use of these techniques in a clinical context is currently illegal, and the ethics of this type of
disease prevention is currently being debated. If the procedure were to be legalized, it would
probably be the first example of germ-line gene transfer in humans.

22