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Gene Mutations

The Place of Genetics in Medicine
• The true scale of genetic disease has
only recently appreciated. • One in 20 people under the age of 25 develop a serious disease with a major genetic component. • Studies of causes of death of more than 1200 British children suggest that 40% died as a result of a genetic condition.


• Some mutation is good, too much is
bad. • Cells employ elaborate mechanisms to prevent mutation, but the mechanism aren’t perfect. • Mutations are the root cause of cancer (bad) • Mutations are the only way to introduce novel genes into a species for evolution (good). • The effects of mutation are usually bad or neutral - only sometimes are mutations beneficial. • So just like Goldilocks - not too hot, not

Gene Mutations & Diseases

• Many, if not most, diseases have
their roots in our genes. • More than 4000 diseases stem from altered genes inherited from one’s mother &/or father. • Common disorders such as heart disease & cancer arise from a complex interplay among multiple genes & between genes and factors in the environment

Gene Mutations
• Genes can be altered or mutated in
many ways • The most common gene change involves a single mismatch (misspelling) placing the wrong base in the DNA • At other times, a single base may be added or dropped • And sometimes, large pieces of DNA are mistakenly repeated or deleted

Classification of Mutations
• Single base substitution (also called
point mutations) – Missense mutation (sickle cell disease) – Nonsense mutation (cystic fibrosis) – Silent mutation (one in G6PD deficiency) – Splice-site mutation (thalassemia) • Insertion & deletions

Mutation Definition

Pu to Pu - transion

uu u


Generation of a 'nonsense' mutation
•  The wild-type DNA sequence 
   5'- CTA CAG ATT - 3'                                    3'- GAT GTC TAA - 5' • Produces the mRNA 5'- CUA CAG AUU - 3'  which codes for the polypeptide  leu gln iso [etc] • A 1st position mutation (C T) in the second triplet gives a 'mutant' DNA sequence    sense strand           5'- CTA TAG ATT - 3'                           3'- GAT ATC TAA - 5' Produces the mRNA which 5'- CUA UAG AUU - 3' codes for the polypeptide leu- *

Generation of an 'amber suppressor' mutation in a tRNA gene

• DNA sense strand  

5'- TAC -3'                             3'- ATG -5'    which produces the tRNA   3'- AUG -5' anticodon loop    which reads the mRNA      5'- UAC -3'    as                             - tyr –

•   A 3rd-position mutation (C- G) in this region
produces a 'mutant' tDNA gene

•   sense strand              

5'-TAG -3'                              3'- ATC -5'   which produces the tRNA    5'- AUC -3'  anticodon loop   which reads the mRNA as  5'- UAG -3'                          - tyr -

Insertions & Deletions
• Each base pairs may be
added(insertions) or removed (deletions) from the DNA of a gene • Insertions & deletions involving one or two base pairs( or multiple thereof)) can have a devastating consequences to the gene because translation of the gene is “ frameshifted” • Insertions or deletions of three nucleotides or multiple of three may be less serious because they preserve the reading frame • A number of inherited diseases are caused by the insertion of many copies


• Duplications are a doubling of a section of
the genome during meiosis • This can cause the new gene to carry inappropriate promoters at its 5’ end


• Transfer of a piece of one chromosome to
a nonhomologous chromosome • This can alter the phenotype in several ways
promoters & enhancers • Breakpoint may occur within a gene creating a

• Break may occur within a gene destroying its function • Gene may come under influence of different

mRNA processing defects Normal splicing






Abnormal splicing

β + thalassemia IVSI-

Frequency of Mutations • Mutations are rare events. • Humans inherit 3 x 109 bp from each parent. i.e.

each cell has 6 billion different bp that can be the target of a substitution. • Substitution occurs when DNA is being copied (during S phase). • No process is 100% accurate. So it is with DNA replication. • Errors occur at a rate of about 1 in every 50 million nucleotides added to the chain. (Not bad, I wish I could type so accurately.) • But with 6 X 109 bp in a human cell, it means that each new cell contains some 120 new mutations. • Should we be worried? Probably not. • Most our DNA does not encode anything. This includes:
– Repetitive DNA(Alu-junk) –introns –synonymous codons

Somatic vs Germline Mutations occur in a somatic cell, • Mutations that

bone marrow or liver, may •Damage the cell •Make the cell cancerous •Kill the cell Whatever the effect, the ultimate fate of that mutation is to disappear when the cell in which it occurred , or its owner , dies • Germline mutations, in contrast, will be found in every descendant from zygote to which that mutant gamete contributed. It will be passed from one generation to the

Males contribute more mutations than Females

• Most mutations occur during S phase of

cell division. Consequently males should be more at risk because: – Only two dozens or so mitotic divisions occur from the fertilized egg that starts embryonic developments – The sperm of a 30 year old man in contrast is the descendant of at least 400 mitotic divisions since the fertilized egg that formed him • The children of aged father suffer more genetic disorders than those of young

Altered DNA-Altered Protein
• A protein encoded by a gene that
contains a mutation is likely to be abnormal. • Sometimes the protein will be able to function, but imperfectly. • In other cases, it will be totally disabled. • The outcome depends not only on how it alters a protein’s function but

Hereditary Mutations • Gene mutations can be either

inherited or acquired • Hereditary mutations are carried in the DNA of the reproductive cells • When productive cells containing mutations combine to produce offspring, the mutation will be in all of the offspring’s body cells

Acquired Mutations
• Acquired mutations are changes in
DNA that develop throughout a person’s lifetime • Although mistakes occur in DNA all the time, a cell has the remarkable ability to fix them • But if the DNA repair mechanism fails, mutations can be passed along to future copies of the altered cell


• Genes come in pairs, with one copy

inherited from each parent. • Many genes come in a number of variant forms, known as alleles. • A dominant allele prevails over a normal allele. • A recessive gene becomes apparent if its counterpart allele on the other chromosome becomes inactivated or lost. • During formation of gametes, the members of each pair separate, so as each gamete carries one allele of each pair. Allele pairs are restored at fertilization. • Alleles of different genes reassort

Altered Dominant Genes
• In a dominant genetic disorder, one
affected parent has a diseasecausing allele that dominates its normal counterpart • Each child in the family has a 50percent chance of inheriting the disease allele and the disorder

Rules for autosomal dominent inheritance

• Both males & females express the allele

& can transmit it equally to children. • Excluding new mutations & nonpenetrance, every affected person has an affected parent. • Direct transmition through three generation is practically diagnostic of a dominant. • In affected families, the ratio of affected to non affected is almost always 3 : 1 • If both parents are unaffected all

• Abnormally short phalanges. • Dominant allele B causes brachydactyly &
every affected person is either BB or Bb. • In practice most heterozygote, because brachydactyly is a rare trait. • Estimation of risk for offspring of a family member can be based simply on the predictions of Mendel’s laws. • However calculations of dominant condition can be problematic since we usually do not know whether an affected offspring is homozygous or heterozygous.

Altered Recessive Genes
• Here both parents, though disease free
themselves, carry one normal allele and one altered allele • Both males & females are affected • Each child has one chance in four of inheriting two altered alleles and developing the disorder, one chance in four of inheriting two normal alleles, and two chances in four of inheriting one normal and one altered allele and being a carrier like both parents • When both parents are affected all the children are affected

• Represent one in 10000 births • They have very pale hair & skin, blue or

pink irides & red pupils • Suffer from photophobia, nystagmus( involuntary eye movements) • Due to deficiency of tyrosinase. • Every albino is a recessive homozygote (aa) and most are born to phenotypically normal parents.

Disease Inheritance is Complex

• Most diseases do not follow

simple patterns of inheritance. • Many factors influence a gene’s ability to build proteins. • Different mutations in the same gene can produce a wide range of effects. • Cystic fibrosis is a good example.

Dominance & Codominance
• • • • •

• Dominance is defined in terms of which two
alternative allele is expressed in the heterozygote. In the ABO blood group system, groups A, B, AB & O are distinguished by whether the RBCs are agglutinated by anti-A or anti-B. RBC of both IA homozygote and IA/IO heterozygotes are agglutinated by anti-B antibody (similar in IB & IBIO). Therefore allele IA & IB are dominant to IO. The RBCs of IO homozygons are not agglutinated by both antibodies (group O). RBCs of group AB individuals carry both A & B antigens. They are agglutinated by both antibodies. Since both expressed, alleles IA & IB are codominant.

Intermediate Inheritance
Incomplete dominance – refers to the situation when gene expression in heterozygote is intermediate between that in the two homozygote. sickle cell anemia. • Example: Heterozygous have both HbA & HbS in their erythrocytes which stay undistorted most of the time. At this level HbA is dominant to HbS. • Under conditions of hypoxia, a proportion do undergo sickling, on this basis HbS is classified as

• Describes the expression of several
different phenotypic features by a single allele. • Most genes have pleiotropic alleles. • An example is Marfan syndrome (tall, long, thin limbs & fingers, susceptibility of aorta to aneurysm (swelling) due to fibrillin -1. • Those that have the disease can vary in severity (variable expressivity)

Sex-Related Inheritance (Recessive) • Rules:
– The incidence of disease is very much higher in males than females. – Mutant allele is passed from an affected man to all his daughters, but they do not express it. – A heterozygous “carrier” woman passes the allele to half of her sons, who express it and half of her daughters who do not. – The mutant allele is never passed from father to son. – Example is G6PD deficiency.

Sex-Related Inheritance (Dominant) • Rules:
– The condition is expressed and transmitted by both sexes. – The condition occurs twice as frequently in females as in males. – An affected man passed the condition to every daughter, but never to a son. – An affected woman passes the condition to every son and half her daughters. – Example: Hypophosphataemia

Mitochondrial Inheritance
• Mitochondrial inheritance reflects the fact
that a zygote receives all its functional mitochondria from the oocyte. • Rules:
– Typically the condition is passed from a mother to all her children. – The condition is never transmitted by men. – Symptoms can vary between mother and offspring, and between sibs, due to heteroplasmy, i.e. variable representation of different mitochondrial populations.

• Example: A form of progressive blindness
called Leber hereditary optic neuropathy


Exceptions to Mendelian Inheritance • X Inactivation: The Lyon Hypothesis

• One of the two X-chromosomes in the
female is functionally inactivated. • The inactivation occurs early in the life of the embryo. • In the embryonic cell, it is a matter of chance as to which of the two Xchromosomes (P or M) is inactivated. • Once an X-chromosome is inactivated in an embryonic cell, all the progeny of that cell maintain the same inactive X. • In individuals with more than two Xchromosomes, all those in excess of one are inactivated.

Female is a Mosaic of Xchromosome gene expression :with

• Patches of cell expressing the genes

of the paternally derived X. • Patches of cell expressing the gene of the maternally derived X. • On average, the split will be 50:50 • Considerable variation could, and in fact does occur because inactivation event is random.

• X-linked recessive disorders. • In many X-linked recessive disorders,

Clinical Implication of X .Inactivation

the heterozygotes have a remarkable range of expression varying from being phenotypically normal to manifesting characteristics of the disorder almost as severely, or as severely as the affected male. • Skewed inactivation (randomness of X-inactivation)

Chemical Mutagenesis
• Environmental
mutagens include constituents of smoke, paints, petrochemicals, pesticides, dyes, food stuffs, drugs etc. • Principle means of exposure are inhalation, skin absorption and ingestion. • Effects include: – Nitrous acid causes transition (C U) – Chemicals which cause DNA strand breakage – Acridine dyes intercalate between adjacent base pair and leads to deletion

Electromagnetic Radiation
• Includes α, β and γ radiation. mutagenicity increases • Their
decreasing wavelength. • All radiations beyond UV causes ionization. Sources include
– Natural background radiation (Potassium 40 and radon 222 gas) – Cosmic rays, major hazards for aircraft as their intensity increases with altitude. – Man-made radiation nuclear testing, power stations. – X-rays


Biological Effects of Radiation ((1 • Electromagnetic radiation damages

proteins and above 100 rads kills cells. • At chromosomal level it causes major deletions, translocation and aneuploidy. • It causes single and double-strand breaks in DNA and base pair destruction. • X-rays damage chromosomes most readily when they are condensed,

Biological Effects of Radiation ((2

• For the first seven days, embryo is
ultrasensitive to the mutagenic and lethal effects of x-rays. Childhood leukemia can be induced by exposure at gestational weeks 8-40. • 100 rads causes 50% drop in WBC count, at 450 rads kills 50% of people, 1000 rads used to kill

Case: A 10 year old male is referred to a genetic evaluation clinic by his pediatrician because of mental retardation.
Labs: All basic & endocrinological lab profiles are normal. Karyotype “ fragile gap” at the end of the long arm on Xchromosome.

Fragile X-Syndrome

• Second most common form of inherited

mental retardation. of trinucleotide repeat • Expansion sequence CGG found on the 5’-untranslated region of FMR1 gene on X chromosome ( > 200 repeat). • This induces extensive methylation of the promoter region & inactivates it, which

• Imbalance in β & α globin peptide
chain synthesis. • Anemia at age of 6 months and requires frequent transfusion. • α-thalassemia is usually due to deletion. • β-thalassemia is due to a wide variety of mutations including frame shift, truncation & splice mutation.

((amanita phalloides

Death CapMushroom
causes several deaths each year. It contains a lethal toxin α-amanitin that inhibits the largest subunit of eukaryotic RNA poly II, thereby inhibiting mRNA synthesis. Initial poisoning is relatively mild – such as gastrointestinal symptoms. This is followed 48h later by massive liver failure as essential mRNAs and their proteins are degraded but not replaced by newly synthesized molecules.

 This is a highly poisonous mushroom that   

A Case of Mistaken Identity((1
An otherwise healthy young man presents himself to the emergency room with severe nausea and diarrhea. His symptoms came on suddenly, about 2-3 hours after he had eaten dinner. The physician suspects some form of food poisoning, and asks the patient to recall everything he has eaten over the past 24 hours. The only suspicious food mentioned were mushrooms that the patient ate for dinner. The mushrooms become prime candidates for the cause of this patient’s symptoms when he further relates that they were picked up on a recent hike through the woods. What is the biochemical basis for suspecting that the

A Case of Mistaken Identity((2
Comment: It is likely that the patient mistakenly picked a member of the family Amanita phalloides and ingested them at dinner. The toxin, α-amanitine, binds preferentially to RNA polymerase II and inhibits its function; if a large quantity of the mushrooms had been ingested, even RNA polymerase III could be inhibited. The first cells that encounter the toxin are those of the digestive tract, leading to acute gastrointestinal distress: cells that are incapable of synthesizing new mRNAs and tRNAs would die, causing the diarrhea and

• Tuberculosis is endemic in
impoverished populations. tuberculosis, the • Mucobacterium causative agent of tuberculosis is insensitive to many commonly used antibiotics, but it is sensitive to rifampicin. produced by soil • Rifampicin, streptomycetes and is an inhibitor of many bacterial RNA polymerases. • Eukaryotic polymerase is not that sensitive.

Systemic Lupus Erythematosus • An autoimmune disease

connective tissue. • Patients exhibit a serum antibody designated Sm. • This serum antibody binds to common core proteins of snRNP (U1, U2, U4, U5) • snRNP are involved in spliceosomes which are responsible for mRNA processing. • Hence antibody inhibits splicing.





• Characterized by polycynthemis (over

• • •

production of RBC due to increase secretion of erthropoietin secreted by the kidneys) Position 145 of the β-chain normally occupied by tyrosine which is coded for by UAU or UAC. Mutations in this codon to UAA or UAG result in termination of β-globin chain synthesis. This gives rise to hemoglobin molecules (144 AA instead of 146)

 Imbalance between α & β-globin chain    

Southeast AsiaβThalassmia

synthesis. β -globin is not synthesized; results in α-globin accumulation and precipitation in erythroid cells. This precipitation damages cell membrane and causes hemolytic anemia. Normal codon 17 in β-gene (AAG) codes for lysine, a mutation leads to UAG (stop codon) which terminates β-globin chain synthesis. The mutation occurs so early in mRNA that no useful β-globin sequence can be synthesized.

Constant Springα -Thalassmia

• This is characterized by abnormally long

α-globin molecule which is unstable & breaks easily. • α-codon 142 (UAA) designates a stop codon with formation of a normal α-globin chain with 141 amino acids. • Mutation to CAA replaces stop codon with codon codes for glutamine (in the untranslated region) • The next stop codon is at 173, hence production of the α-globin with 172 amino

Antibiotic-Induced Deafness
• In some regions of China, use of gentamicin & • • • • •
streptomycin leads to deafness. This is a maternally transmitted sensitivity which is therefore due to mitochondrial DNA. Codon 1555 of the gene on mitochondrial DNA codes for the rDNA of the A-site of the large subunit. A mutation A to G results in making the region more prokaryote-like, increasing its affinity for aminoglycosides binding Antibiotc bindings therefore interfere in protein synthesis in the mitochondria. This antibiotic tends to accumulate in the cochlea, making this a particularly sensitive target and leading to sensorineural deafness.

Ehlers-Danlos Syndrome
• Case: A 9-year-old boy is brought to ER with pain,
inability to lift his left shoulder and flattening of the normal rounded shoulder contour (Shoulder dislocation) that occurred when he tried to hit a ball with a baseball bat. PH: He dislocated his left shoulder nine times before and his right shoulder three times. He also has a history of easy bruising and arterial rupture. PE: Hyperelastic skin, hyperextensitivity of joints. Micropathology: Collagen fibrils of dermis skin larger than normal and irregular in outline. Discussion: Genetic disease, autosomal dominant or recessive (10 types). Due to point mutation in a code for glycine and thus disruption of the collagen triple helix. It can also be due to the exon skipping which shortens the polypeptide.

• • • •

Familial Hyperinsulinemia
• Autosomal dominant condition which is
characterized by mild diabetes. • Approximately equal amounts of insulin and an abnormally processed proinsulin is released into the circulation. enzymes process proinsulin; • Three endopeptidase that cleaves the Arg31Arg32 & Lys64-Arg65 peptide bonds and carboxypeptidase. • Defect is due to substitution of Arg65 by HIS or LEU which prevents the cleavage between the C-peptide and the A chain of insulin resulting in secretion of a partially processed proinsulin.

Glycosylation of proteins to form glycoprotein is important for two reasons: 3. Glycosylation alters the properties of proteins changing their stability & physical bulk. 4. Carbohydrates of proteins act as recognition signals that are central to aspects of protein targeting and for cellular recognition of proteins

Glycosylation of Proteins by ER

I-Cell Disease ((Mucolipidosis • Defects in lysosomal

enzyme targeting due to deficiency of N-acelylglucosamine phosphate transferase. • Cells show dense inclusion bodies and the enzymes are secreted in the medium. • Patients have high level of lysosomal enzymes in their sera. • Characterized by: – Severe psychomotor retardation – Skeletal deformaties – Coarse facial features – Restricted joint movement – Death by age of 8

(Trinucleotide Repeats (1
Case: A thirty-three year old woman has recently been diagnosed as having mild myotonic dystrophy (MD). MD is characterized by autosomal dominant inheritance, muscular weakness associated with impaired muscular relaxation, frontal balding, endocrine disorders such as diabetes and premature ovarian failure, and premature cataract formation. The patient has muscular problems and frontal balding, whereas her affected father, aged 60 years, has only just developed cataracts. She is concerned about the risk to her own children if she becomes pregnant.

(Trinucleotide Repeats (2

• The majority of nucleotide repeats are non-coding

• • • •

(clinically silent), however several are related to human disease (unstable). Unstable repeats undergo changes in size (expansion) during meiotic division where during each successive meiosis, expansion of the repeats will gradually increase in size (anticipation – progressive worsening of the clinical phenotype with successive meioses). Once trinucleotide expansions reach a critical size, they begin to interfere with gene function and thus result in the clinical syndrome. In the case of MD, with an expansion size of 80 repeats, the patient will exhibit signs of the disease. Owing to instability of the triplet repeat during meiosis, offspring of this patient might expect to have a larger expansion and also display features of the disease at a younger age. Anticipation may be sex limited, with the largest expansions only present in eggs, which have larger amounts of cytoplasm than the spermatozoa

Protein Truncation Testing in Breast Cancer

• A hereditary form of breast cancer associated • •

with coexistence of ovarian cancer has been shown to be due to mutations in the BRCA1 & 2 gene (tumor suppressor gene). Most of the mutations were either nonsense mutations, altered splice sites, or frameshift mutations which give rise to abnormally truncated protein products. Screening for mutations using a protein truncation test has become standard practice when examining both pathologic tissue and blood from individuals at risk of possessing a BRCA 1 or BRCA 2 mutation. Comment: The translation of mRNA to the final protein product results in a peptide of predictable size. If a gene has a mutation that results in a premature stop codon, then the resulting peptide will be abnormally small.

Reverse transcriptase-PCR

From whole blood, or pathologic tissue, cDNA complimentary to the mRNA for a gene can be prepared by the process of reverse transcription (reverse transcriptase). RT, using a single poly T primer, recognizes the mRNA via its poly A tail. The resulting cDNA is then used as a template for a PCR where the entire cDNA can be amplified to produce a DNA molecule containing the entire coding sequence of a gene.

Reverse transcriptasePCR((2
Clearly in some cases, gene expression is issue-specific and one would not expect some mRNAs to be present in blood, e.g. dystrophin from muscle. However, ectopic transcription of genes occurs in white blood cells at a low level and allows analysis of transcripts of genes not normally expressed. This can be useful if the desired transcript is derived from a tissue that is not easily accessible or if study of the cDNA can give definite information about the presence of a mutation.

A Noncompliant Patient (1)
A young man you were treating for a sinus infection returns to your clinic after 1 week, still complaining of sinus headaches and stuffiness. He explains that he began to feel better about three days after starting to take the antibiotic tetracycline, which you had prescribed. You inquire as to whether he continued to take the full dose of the drug, even after he began to feel better. He reluctantly admits that, as soon as he felt better, he stopped taking the drug. How do you explain to your patient that it is important that he takes the drug for as long as you prescribed it, even if he feels better after only a few days?

A Noncompliant Patient (2)
As a physician, you know that tetracycline inhibits the protein synthetic machinery of the bacterial cell by binding to the A site of the ribosome. You also know that, if the drug is removed, protein synthesis can resume. If the drug is not taken for the entire period recommended, bacteria will begin to grow again, leading to the resurgence of the infection. Further, those bacteria that begin to grow after early termination of treatment are likely to be the most resistant to the drug, either because of the selection of more resistant strains, or because of mutation to more resistant strains. The secondary infection is therefore likely to be more difficult to control.

(The Case of a Stubborn Microbe (1 A patient you were treating for an infected wound last week returns to your clinic, complaining that the infection is worse. You examine the wound and confirm that indeed the infection has become worse, even though you had prescribed a highdose regimen of antibiotics that targeted the bacterial protein synthetic machinery. After questioning the patient to ensure that she was compliant and took the medication, you elect to prescribe rifampicin, a synthetic derivative of the naturally occurring antibiotic, rifamycin. The patient asks you why you think this antibiotic will work.

(The Case of a Stubborn Microbe (2

Comment: Antibiotics work by targeting specific functions in the bacterial cell. In the first round of treatment, you used antibiotics that inhibited the bacterial protein synthetic machinery. In some cases, microbes become resistant to a specific type of antibiotic and it is necessary to target other bacterial functions in order to clear up the infection. Rifampicin inhibits the transcriptional machinery of bacteria, inhibiting the β subunit of bacterial RNA polymerase. Without the ability to make RNA, the bacterial cell will die.

(Therapeutic Application of Ribozymes ( HIV

• They have the potential to become useful

therapeutic agents. • Inhibitors of viral gene expression such as HIV using Ribozyme have the following advantages over other strategies.
– 1. Cleavage of viral transcripts results in direct irreversible inactivation of the target RNA. – 2. Few ribozymes may be required to inhibit a given target gene effectively because a single ribozyme can catalyze multiple cleavage reactions and thus destroy multiple viral transcripts. (Note – Ribozyme may also be sensitive to HIV sequence heterogeneity)

(Therapeutic Application of Ribozyme ( Oncogenes

• Neoplastic

transformation is often associated with the expresion of mutant oncogenes. • Ribozymes can be designed to inhibit the expression of specific gene product. • It has been reported that hammerhead ribozymes are able to suppress the tumorigenic properties of cell harboring an activated human ras gene. leukemia and chronic • Lymphcytic myelogenous leukemia express the transformation of an abnormal mRNA, this transcript has been shown to be cleared

Antisense Oligonucleotide • Antisense oligonucleotides are short synthetic olionucleotides (15-25 bases). • Designed to hybridize to mRNA through watson-crick base pairing. • Upon binding to target RNA, the oligonucleotide prevent expression of the encoded protein product in a sequence specific manner. • Currently one antisense product on the market used in therapy.

• This requires two factors:
1. Introduction of a gene for an enzyme which is not expressed by the mammalian cell. 2. A non-toxic pro-drug which can be converted into a toxic metabolite.

Suicide Gene Therapy

• Most widely used suicide gene is the
herpes simplex virus thymidine kinase (HSV-tk) gene, used with the pro-drug Ganciclover.

HSV-tk Suicide Gene
• This strategy is effective against solid tumors. It is • • •
generally less effective against hematopoietic malignancies. tk enzyme is foreign to mammalian cells and converts nonactive ganciclovir into a toxic product (monophosphate form). Intracellular host kinases convert the monophosphate into di and tri phosphate form. Triphosphate form is incorporated into the replicating DNA chain and inhibits DNA polymerase which results in replication termination and cell death. Bystander effect is also seen, where neighboring normal cells are also effective due to exchange of toxic metabolites.

Disadvantage of Suicide Gene Therapy

• It requires S-phase cell cycle activity
and thus targets only dividing cells
(not all malignant cells within a tumor are cycling but different rates of cell doubling is seen and this requires a longer time of gene expression)

• Although bystander effects help

augment the anti-tumor effects, there is a likely threshold of HSV-tk transfer and expression and persistence of the enzyme which must be achieved to generate or sustain a significant therapeutic effect.

Cytosinedeaminase System
• This enzyme is found in fungi and is
not expressed in mammalian cells. It catalyzes deamination of cytosine into uracil. • It can also convert pro-drug 5flurocytosine into the metabolite 5flurouracil. • Intracellular enzymes convert 5flurouracil into 5-flurouridine-5triphosphate and 5-fluro-2deoxyuridine-5-phosphate, both of

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