Biochemistry Review for Section B, Pages 31-55
Classifications of DNA mutations
1. Cytogenic
2. Molecular
a. single base
1. missense-mutation causes AA difference.
2. Nonsense-mutation results in early termination codon, resulting in
truncated protein.
3. splice site-mutation results in altering of intron splicing, can cause a
frameshift mutation.
4. conservative-usually in the 3rd codon. Does not affect AA.
b. Length variation
1. deletion-deletes a base, results in frameshift mutation.
2. duplication-duplicates a sequence
3. insertion-inserts an extra base, can lead to frameshift mutation
4. Expansion-??
Triplet repeat expansions
1. Lead to neurogentic diseases and are always caused by an increase in the
number of normal triple repeats.
2. Typically seen in noncoding DNA, with the exception of the CGA repeat in
coding DNA that codes “toxic polyglutamine tracts.”
3. Severity proportional to the size of the repeat length.
4. Leads to anticipation – increase in severity with successive generations.
5. Diagnosed by PCR or Southern blot.
6. Examples – Fragile X-syndrome, Huntington Disease, Friedrich Ataxia,
Myotonic Dystrophy
7. Premutations are triplet repeat expansions consisting of 20-40 repeats and
may lead to the development of a mutant allele.
Detection of mutations
1. PCR is used for small mutations (<1kb), while southern blot is used for
larger mutations (>1kb).
2. Different strategies are employed to detect mutations that are known vs.
mutations that are unknown.
a. When the mutation has previously been identified, say in another
family member or by other means, you can screen for that mutation by
itself. This can be done by a restriction digest, where the mutation
can be detected by the creation or disappearance of a restriction site. If
this is not possible then a dot blot can be performed with allele
specific oligonucleotides (ASO). These are sequences about 20 bp
long that are created to compliment the allele in question (with the
known mutation).
b. If the mutation is unknown there are 5 methods of detection
1. Southern blot, hybridize with cDNA probe – this is the only way to
detect major deletions and rearrangements. Large quantities of DNA
are required and the process is laborious and expensive.
2. Heteroduplex analysis – This method involves introducing to the
unknown DNA mixture, normal DNA of the same sequence. The
strands are denatured and then reannealed. The mixture is run on a gel
and if a mutation is present, the gel will show multiple bands.
Drawbacks to this method include limited sensitivity, small sequences
(<200 bp), and it obviously does not reveal the position of the
mutation.
3. Single stranded conformation polymorphism analysis - ?
4. Chemical cleavage of mismatches - ?
5. Sequencing – Is the “gold standard” in detecting mutations. Due to
the low cost of sequencing some investigators will sequence entire
genes and analyze for mutations instead of scanning for mutations.
Sequencing provides exact information on the mutation.
Causes of mutations
1. Mutations are caused in four ways
a. Faulty DNA replication during cell division.
b. Spontaneous deamination of methylated CpG dinucleotides – This
dinucleotide is subject to 8.5 times higher mutation because normal
deamination of cytosine to uracil is recognized by the repair system.
When cytosine is methylated and then deaminated it is not recognized
by the DNA repair system because it has effectively been converted to
a normal DNA base.
c. Spontaneous chemical attack – Usually in the form of active oxygen
species that cause massive depurination (at a rate of 5000 adenines and
guanines a day).
d. Mutagens – Such as UV light and radiation.
2. Mutator phenotype – when a cell develops multiple mutations due to defects
in DNA repair mechanisms.
3. Examples of DNA repair mechanisms
a. Base excision repair – removal of abnormal bases in DNA.
b. Nucleotide excision repair – removes thymine dimmers caused by
UV exposure.
c. Transcription coupled repair – corrects errors in transcribed genes.
d. Post replication repair – recombines dsDNA breaks caused by Xrays
and anticancer drugs.
e. Mismatch repair – corrects mismatched base pairs.
Identification of a diseased gene – there are 2 methods that are employed to determine a
diseased gene
1. Functional cloning – This can be performed by two methods
a. First the protein that is causing the disease is identified. The protein is
partially sequenced and this sequence allows for creation of a cDNA
probe. DNA libraries are searched with the probe and the gene
responsible is identified.
b. Or, you can create an antibody to a given protein by injecting animals
with the foreign protein. The animal’s immune system will create
antibodies to this protein and those are purified. Screen an expression
cDNA library against the antiobody to identify the gene.
2. Positional cloning – You first map the location of the gene by linkage
analysis. You can then create a protein from the gene and determine the
function of that protein.
a. Linkage analysis is a process by which polymorphisms are used to
determine the location of gene. This is possible because the HGP has
determined the location of many polymorphisms and distance to a
gene on a chromosome is a function of the frequency of crossing over
between the polymorphism and the gene of interest. Crossing over
occurs in meiosis I when bivalents (chromosomes paired with one
another) contact one another (synaptomeal complexes) and separate
causing recombination. Genetic distances are measured in
centimorgans where 1% frequency of recombination occurs.
b. Problems with positional cloning can arise in two ways
i. Allelic heterogeneity – where a mutation in one location can
cause several diseases.
ii. Locus heterogeneity – where defects in the same gene causes
different diseases.
Human gene project – was started in 1990 and was expected to be completed in 2003.
1. The human draft was completed by shotgun sequencing which is creating a
HG BAC library and then subcloning each BAC into smaller clones. The
smaller clones are assembled and analyzed by computers.
2. Also other model organisms were sequenced including yeast, worms, fruit
flies, chimpanzees, mice, rats, and dogs.
3. Benefits of the human genome project include…
a. Disease gene ID
b. Gentic diagnosis
c. Pharmacogenics
d. Succeptibility markers for complex diseases
e. New genetic functions and pathways
4. ELSI (ethical, legal, and social implications)
a. Privacy and fairness including insurance, employment, and
educational.
b. Clinical integration of new genetic technology
i. Informed consent
ii. Predispositional counseling
iii. Cultural differences
c. Genetic research – availabitity
d. Education of health care professionals and the public
Genetic disease, counseling, and therapy
1. Ways to control genetic disease
a. Population screening
i. Examples are 95% decrease in the incidence of Tay-Sachs
disease among Ashkenazi Jews and 95% decrease in beta
thalesimia in Italians.
2.
Requirements for implementing genetic screening programs
a. Positive result must lead to useful action
b. Program must be socially and ethically acceptable
c. Tests must have high sensitivity and specificity
i. Sensitivity – the frequency that a test is positive in people with
disease.
ii. Specificity – the frequency that a test is negative in people
without the disease.
d. The benefits must outweigh the costs.
3. Oklahoma tests for PKU, galactosemia, congenital hypothyroidism, and
sickle cell disease.
Genetic counseling – the major focus is on prevention and avoidance
1. Family history is the most important and useful tool.
2. The physician must communicate the medical facts, risk of recurrence,
options for courses of action, and the adjustments that family will have to
make about the disease.
3. The physician must not pressure the decision of the family.
Indications for prenatal diagnosis
1. Mothers that are over 35 years old.
2. A family that has already had a child with a defect, family history.
3. Parents that have been diagnosed with a genetic abnormality.
4. abnormal maternal serum for alpha feto-protein test
5. Abnormal maternal serum in triple marker screening test.
Amniocentesis vs. chorion villus sampling
1. Amniocentesis is the removal of amniotic fluid at 16 weeks. This can test
for alpha feto protein levels that signal neural tube defects. Two positive
results in 18-20 weeks gestation time indicate a 1/20 chance that the fetus
has a NTD. This is not however diagnostic. Ultrasound is the diagnosis
method. The drawbacks of this method are that it is done later in the
pregnancy and the results can take a long time to get back.
2. Chorion Villus sampling – this is the suction of chorion fundosum (the
precursor of the placenta). The test can be done at 9-12 weeks and this is
one advantage over amniocentesis. CV sampling cannot detect NTD’s.
Allows for analysis of DNA.
3. Triple test marker – tests for an increase in MSAFP, increase in HcG,
and a decrease in unconjugated esterdiol. This test is used to detect 50-6% of all trisomy 21 babies in mothers over the age of 35.
Gene therapy – gene therapy is still experimental and involves gene augmentation
which is the delivery of a normal gene into the patient. This is only done in somatic cells
(vs. germline cells) There are two types of somatic gene therapy
1. in vivo – in this method the normal gene is delivered directly into the body,
usually by exploiting the ability of viruses to replicate DNA inside the host, or
by coating the gene with liposomes which facilitate intracellular delivery.
2. ex vivo – in this method cells are extracted from the patient, manipulated with
the normal gene and then reintroduced into the patient.
Genetics of multifactorial diseases (polygenetic)
1. Usually the traits are quantitative (i.e. blood pressure) and continuous
(normal in the population)
2. QTL – quantitative trait loci – is the loci that express a polygenetic trait.
And when the disease is present it is called a disease susceptibility locus.
3. Genetic relationship between relatives is an important diagnostic tool in
determining a patient’s susceptibility of a polygenetic disease.
Twin Studies – are used to determine if a disease in caused by the environment, genetics,
or a combination of the two
1. Important terms
a. Concordance – When both twins either have or don’t have the
disease.
b. Discordance – When one twin has the disease and the other doesn’t.
2. 100% genetic disease – in this type of disease there is 100% monozygotic
twins (MZ) concordance and 50% of dizygotic twins (DZ) have the disease.
3. 100% non-genetic diseases (environmental) – MZ and DZ twins have equal
concordance.
4. Multifactorial diseases – MZ twin concordance in less than 100%, but is
significantly higher than DZ twins.
5. recurrence risk of multifactorial genes is dependent on several factors
a. closeness of affected relatives
b. sex
c. disease severity
d. the number of affected relatives
Hardy Weinberg equilibrium – can be used to calculate the carrier frequencies and the
simple risks for counseling
1. For HW to work certain factors must be true, these are…
a. There must be random mating in a population
b. The population must be large
c. There must be no mutations
d. There must be no genetic drift
2. For an autosomal locus with 2 alleles A1 and A2 with frequencies of p and q,
the chance that individuals will be homozygous for A1 is p2, the chance that
an individual will be homozygous for A2 is q2, and the chance that an
individual will be heterozygous in 2pq.
3. For an X chromosomal locus with alleles A1 and A2 with frequencies of p
and q again, the chance of females being homozygous for A1 is p2. The
chance that a female will be homozygous for A2 is q2, and the chance that a
female will be heterozygous is 2pq. The chance that a male will have
genotype A1 is p, and the chance that a male will have genotype A2 is q.
Ethical considerations in medical genetics
1. Individuals have the right to be in control
2. Individuals cannot be pressured into making a decision.
3. Privacy and confidentiality must have utmost importance.
4. Losses of jobs, or being dropped from insurance coverage are equity issues.