MS-1 FUND 1: 9:10-10:00
Friday, September 12, 2014
Dr. Korf
Genetic Testing
Transcriber: Lizzie Ernstberger
Editor: David Woods
Page 1 of 4
Abbreviations: FISH- Fluorescent in situ hybridization; CF- Cystic Fibrosis
Introductory Comments: This is the hour-long lecture on genetic testing given by Dr. Korf. Audio times are from the
ECHO recording.
ARS question about genetic testing 0:39
I.
Indications for Genetic Testing (slide 4) 3:59-5:20
a. Genetic testing requires a blood sample to extract the DNA—the DNA does not have to be presently expressed
for the condition to be detectable, as the genetic information is still present in the sequence. This is much less
invasive than previous procedures that required muscle biopsies (Muscular Dystrophy, eg.)
i. Five indications for genetic testing:
1. Establish a diagnosis in a symptomatic individual
2. Obtain a prenatal diagnosis
3. Diagnose an at-risk individual that is pre-symptomatic
4. Test individuals for carrier status
5. Perform predispositional tests for common diseases
II. Copy Number Genetic Tests (Cytogenetics and Cytogenomics) (slides 5-6) 5:20-13:25
a. Copy number mutations refer to an improper amount of a gene resulting from duplications (too much) or
deletions (not enough)
i. Cytogenetics 5:20-9:25
1. Examining genetic mutations at the chromosome level
2. Has evolved since the early 1950s to include fluorescent tracking of particular genes (FISH)
a. Label the DNA sequence of interest with a fluorescent marker and track it to the
chromosome—if it fails to light up, the gene is missing and the mutation is a deletion
3. Clinical example: Trisomy 21
a. Trisomy 21 (Downs Syndrome) was the first disease to be indentified cytogenetically
(1950s)—identified the extra copy of chromosome 21
b. Allowed researchers to track the distinct phenotype seen in Trisomy 21 to the genetic level
c. Trisomy is only capable of producing viable individuals on chromosomes 13, 18, and 21 due
to their small and gene sparse nature
4. Disadvantage: geneticists have to know exactly what region of DNA they are looking for
ii. Cytogenomics 9:25-13:25
1. Allows geneticists to detect small increases or decreases in DNA content on a chromosome on
levels far below what is visible on a microscopy
2. Process of cytogenomic testing:
a. More advanced technique
b. Obtain a DNA sample from the patient’s blood and fragment the DNA
c. Label the fragments with a fluorescent dye (red dye = patient DNA)
d. Combine the patient’s single-stranded DNA with fluorescently labeled, single-stranded
reference DNA (green dye = reference DNA)
e. Single strands will bind together based on homologous sequences
f. DNA is placed on a chip and the resultant colors are analyzed:
i. Green spot- deletion, patient does not have enough DNA content, so reference DNA
dye color is prominent
ii. Red spot- duplication, patient has too much DNA content, so patient DNA dye color is
prominent
iii. Yellow spot- proper amount of DNA content in the patient sample, so colors blend to
produce a yellow spot
3. Benefit: do not have to know what gene sequence you are looking for (in contrast to
FISH/cytogenetic testing)
III. Spectrum of Molecular Disorder Genetic Tests (slides 7-10) 13:25-14:25
a. Three types: targeted mutation, mutation hot spots, and diverse mutations
i. Each of these tests can identify mutations in a single base pair
b. Targeted testing: Sickle Cell Anemia 14:25-15:51
i. Sickle cell disease is always based on a single base substitution—targeted testing relies on restriction
endonucleases that can recognize variations in specific base sequences
1. The restriction endonuclease (ex. MSTII for sickle cell) will only cut a fragment of DNA if the
sequence is normal (base = A) and will leave the fragment uncut if it codes for sickle cell (base = T)
MS-1 FUND 1: 9:10-10:00
Friday, September 12, 2014
Dr. Korf
Genetic Testing
Transcriber: Lizzie Ernstberger
Editor: David Woods
Page 2 of 4
Abbreviations: FISH- Fluorescent in situ hybridization; CF- Cystic Fibrosis
a. Homozygous for mutation: one uncut band
b. Homozygous for wild type (normal): two bands cut at the potential mutation point
c. Heterozygous: three bands (two pieces from wild type endonuclease activity + one uncut
band from mutated sequence)
c. Mutation Hot Spots: Cystic Fibrosis 15:51-18:46
i. Not all diseases are the result of a single mutation—cystic fibrosis, for example, can be caused by a
number of different mutations
1. No one genetic test will pick up all possible mutations, important to test for “hot spots” or common
mutations that produce the specific phenotype
2. Most common mutation for CF (found in over 70% of patients) = deletion of Ph508
ii. Background information on CF: autosomal recessive disorder that produces unusually thick secretions
(result of a deficient chloride ion channel)—becomes especially problematic in the lungs, pancreas, and
small-intestine
d. Diverse Mutations: Neurofibromatosis I 18:46-20:46
i. Just need to appreciate how complicated this process is and why only few labs (like UAB) are able to
perform this type of testing
IV. Prenatal Diagnosis (slide 11) 20:46-23:16
a. Usually performed because an individual is at-risk (based on family history)
i. Amniocentesis: fetal tissue is obtained from the fluid of the amniotic sac, safe procedure usually
performed at 16-18 weeks of the second trimester
ii. Chorionic Villus Biopsy: placenta of fetal origin is obtained through a catheter or needle, performed at
approximately 10-12 weeks
iii. Pre-implantation diagnosis: used in conjunction with in vitro fertilization prior to implantation
1. Single cell biopsy taken from the pre-implanted blastomere and sequenced to discover if the embryo
has the mutation
2. Removing one of the 8 cells of the blastomere does not hinder the embryo’s viability
V. Carrier Testing (slide 12) 23:16-26:23)
a. Parents often discover they are carriers of a disease by conceiving a child with the phenotype
b. Carrier testing is used to screen people with a known genetic risk so they can use that information for family
planning purposes
i. Especially important in certain ethnic groups with high risk (Ashkenazi Jewish population and Tay-Sachs,
eg.)
ii. Common carrier screening diseases include: Cystic Fibrosis, Tay-Sachs, Hemoglobinopathies, Gaucher’s
Disease, Canavan Disease
VI. Pre-symptomatic Testing (slides 13-15)
a. Diagnosing a condition of adult onset before any symptoms occur
b. Huntington’s Disease: 26:23-28:21
i. If an individual has inherited the gene, they are basically guaranteed to become symptomatic at some point
in their life
ii. Because there are limited treatment options and no cure, some people would rather not receive this
information (ticking time bomb analogy)
c. BRCA I and BRCA II 28:21-28:29
i. Having the genes that predispose an individual to breast cancer (BRCA I) and ovarian cancer (BRCA II)
does not guarantee that an individual will get cancer, though there is a substantial risk (80% chance for
breast cancer; 40% for ovarian cancer)
ii. There are treatment and prevention options available, including: imaging surveillance, preemptive
chemotherapy agents, surgery, and self-examination
d. Factor V Leiden 29:29-32:30
i. Factor V is associated with blood clotting—Factor V mutations make an individual immune to protein C, an
enzyme responsible for deactivating the clotting activity of Factor V
1. Mutations result in uncontrollable clotting and potentially lethal thromboses
ii. Only treatment option is preventative blood thinners (which have substantial risks)—based on the
cost/benefit analysis, Factor V Leiden testing is only given to those with a demonstrated, present risk of
clotting
VII. Interpretation of Genetic Tests (slides 16-21) 33:30-39:30
a. Analytic Validity
MS-1 FUND 1: 9:10-10:00
Friday, September 12, 2014
Dr. Korf
Genetic Testing
Transcriber: Lizzie Ernstberger
Editor: David Woods
Page 3 of 4
Abbreviations: FISH- Fluorescent in situ hybridization; CF- Cystic Fibrosis
i. Is the test result correct? Test results are generally accurate
b. Clinical Validity (slide 17)
SN: the slide organization starts to jump around in terms of the clinical examples—the transcript text has been
grouped by topic for this section but does not always follow the chronological order of the presentation—see slide
numbers within the subsection for reference
i. Does the test accurately predict the disease?
1. Positive predictive value:
a. Benign genetic variant vs. pathogenic mutation
i. Ex: molecular diagnosis of Tuberous Sclerosis (slides 18-19)—identified a genetic
variant (missense mutation) in a family that was previously undocumented—geneticists
had to determine if this familial variant (found in patient, sister, and in father) was the
pathogenic origin of the phenotype
ii. Eventually determined that a new stop-mutation was responsible for the child’s
phenotype and that the variant found in the sister and father was not pathogenic
b. Will the phenotype actually present, or is it a case of incomplete penetrance?
i. Ex: Hemochromatosis (slide 21)—autosomal recessive disorder, good example of nonpenetrance
ii. Hemochromatosis is a disorder of iron absorption and fairly common in Caucasians
(1/400) and even higher in Celtic populations (carrier frequency = 1/10)
iii. Even with the homozygous gene for improper iron absorption, only 50% of males
express the phenotype (even smaller proportion in females)
2. Negative predictive value:
a. Incomplete ascertainment of mutations
i. Negative result does not guarantee the patient is free of the mutation
b. Genetic heterogeneity
c. Mosaicism
i. Some mutations are only found in specific cells and would not necessarily be evident in
a blood sample
3. Evidence of Pathogenicity: (slide 20)
a. What is the effect on protein expression?
b. Is this mutation seen only in affected individuals?
c. Does this mutation segregate into disease within families?
d. Is this mutation genetically conserved?
c. Clinical Utility
i. Does the test result make any difference?
d. Ethical Issues
i. Does the test result place the individual at risk for negative ethical or social outcomes?
VIII. Social Issues in Genetic Testing (slides 22-25) 39:30-42:30
a. Genetic Discrimination (slide 22-23)
i. Based on the Genetic Nondiscrimination Act of 2008, it is illegal to use genetic information for employment
decisions or health insurance decisions
1. This protection does not apply to life insurance or disability insurance
b. Gene Patenting
i. Recent history of litigation involving “ownership” of gene sequences—restrictions on where sequencing can
be performed have been removed by the courts
c. When to Test Children
i. Based on a concern for the best interest of the child, physically and psychologically
ii. Have to consider privacy interests and social development
iii. Only test the child if there are clear and immediate medical benefits to the information
1. Defer testing if there are no benefits until adulthood, if the disease is adult-onset only, or if carrier
status is the primary concern
IX. The Future of Genetic Testing (slides 25-30) 42:30-50:40
SN: Dr. Korf very briefly touched on these subjects and said that the technology information is less important to focus on
a. Massively Parallel “Next Generation” Sequencing
i. sequencing now requires a fraction of the original cost, time, and equipment
b. Genome vs. Exome sequencing (slides 27-38)
i. Only testing approximately 1.5% of entire genome, as this is the area that largely codes for proteins
MS-1 FUND 1: 9:10-10:00
Friday, September 12, 2014
Dr. Korf
Genetic Testing
Transcriber: Lizzie Ernstberger
Editor: David Woods
Page 4 of 4
Abbreviations: FISH- Fluorescent in situ hybridization; CF- Cystic Fibrosis
c. Gene Discovery
i. A process of elimination and comparison with sets of siblings within three families
ii. Operated under the assumption that if a condition was rare, a rare trait had to be responsible—excluded all
common sequences and then narrowed down areas of commonality and overlap between patients
d. The Incidentalome
i. When analyzing an entire genome, there is a high risk of discovering other areas of mutation that are not
relevant to the condition at hand—raises ethical issues in terms of disclosing these “incidental” findings
No student questions.
<END OF LECTURE 51:40>