Exam 2 - Genetic testing and Gene therapy Flashcards
Genetic testing and Gene therapy
Goal of screening
Early identification of a disorder so that intervention can prevent or reverse the disease process
Assist in reproductive decisions
Determines the genotypes in individuals that:
1) Already have a disease or predisposed to a disease
e. g. newborn screening
2) Are presymptomatic for a disease
e. g. Huntington disease, breast cancer, colon cancer
3) May have a disease genes that could be passed down to descendants
e. g. Fragile X gene in carrier females
Parameters for genetic screening tests:
Validity
Specificity
Sensitivity
Validity
Refers to the ability of a test to separate individuals who have the disease from those who do not.
Validity involves 2 components: sensitivity and specificity
Sensitivity
the ability to correctly identify those with the disease . It is measured as the proportion of affected individuals who test positive (i.e. true positives)
Specificity
ability to correctly identify those without the disease. It is measured as the proportion of unaffected individuals in whom the test is negative (i.e. true negatives)
_____ and _____ are determined by comparing the screening results with those of a _____ diagnostic test
Sensitivity
specificity
definitive
Test result vs true status
(true status)
+ -
(test) + A B
- C D
Sensitivity (calc)
a/a+c
Probability that test is positive when person actually has the disease
Specificity (calc)
d/b+d
Probability that test is negative when person truly does NOT has the disease
False Positive
b/b+d
Test reports positive for person who does NOT have the disease
False Negative
c/a+c
Test reports negative for person who actually has the disease
High ____ value results in high ____
“a”
sensitivity
High ____ value results in high ____
“d”
specificity
PPV
Positive Predictive value
Positive Predictive value
- Proportion of positive test results that are truly positive
- answers the question: if my test is positive, what is the chance that I am truly positive for the condition?
- a/a+b
NPV
Negative Predictive value
Negative Predictive value
Proportion of negative test results that are truly negative
Answers the question: if my test is negative, what is the chance that I am truly unaffected??
d/c+d
____ of a test varies based on _____ of the condition in a given pop
PPV
prevalence
The ____ the condition in a given pop, lower the ____
rarer
PPV
More detailed PPV calculation
Sensitivity x prevalence
(divided by)
(sensitivity x prevalence) + (1-specificy) (1-prevalence)
In trying to cast a wider net to catch more patients, the trade off is having __________
increased false-positive rate
Screening tests are never 100% ____ and 100% ____.
sensitive
specific
non- invasive pre-natal screening methods:
Ultrasound Nuchal Translucency Index Maternal Serum screening Triple screen; quad screen Fetal cells Cell free fetal DNA
Invasive pre-natal diagnostic methods:
Amniocentesis
Chorionic Villus Sampling (CVS)
Percutaneous Umbilical Blood Sampling
Ultrasound
Can be used to determine presence of spina bifida (meningomyelocele) displayed as a fluid filled sac in the spinal column
Nuchal Translucency Index
Uses ultrasound to measure the translucent space in the tissue at the back of your developing baby’s neck.
Nuchal Translucency Index used to determine:
baby’s risk for Down syndrome (DS) and other chromosomal abnormalities, as well as major congenital heart problems.
Babies with abnormalities tend to accumulate more:
fluid at the back of their neck during the first trimester. Result is an enlargement in this region
US vs Sonogram
US - the machine that allows you to see your baby during your pregnancy
Sonogram - the picture taken of your baby during the ultrasound.
______ take _____ images, or 3D sonograms, and adds the element of time to the process.
4D Ultrasounds
3D Ultrasound
In prenatal screening, confirmation of a _____ result should be followed up with _____
positive
diagnostic testing
Amniocentesis
20-30 mL of amniotic fluid is withdrawn transabdominally with US guidance
Amniocentesis usually occurs:
15-17 weeks gestation
CVS procedure:
a catheter is inserted through the cervix and several mg of villus tissue is suctioned with US guidance
Fetal treatment is _____
rare
Fetal treatment used in the following 3 conditions:
Congenital Adrenal Hyperplasia
Biotin-responsive Multiple Carboxylase Deficiency
fetal surgery for a neural tube disorder
In NBS, _____ screen results need to be followed up with further _______ that is specific for _________
Positive
confirmation testing
each condition identified
Methods of DNA analysis of biosamples:
PCR
ASO (allele specific oligonucleotide) using dot blot
DNA sequencing
Many other methods
allele specific oligonucleotide used frequently for:
Sickle cell and cystic fibrosis
_________ Is the UltimateWay to Characterize a Clone
DNA Sequencing
DNA that you saturate the filter paper with (ASO method)
Probe
Antisense strand
Sample DNA from patient that complements either prode
Based off how the hybridization occurs in ASO:
You can tell whether patient is homozygous normal, a carrier, or diseased
Newer methods of sequencing associated with Next Generation Sequencing (NGS):
Pyrophosphate sequencing
DNA sequencing by the _______ developed by _____
chain termination method
Sanger
Pyrophosphate sequencing relies on the “___________” principle rather than chain termination
sequencing by synthesis
Pyrophosphate sequencing step 1:
ss DNA template is immobilized, and solutions of A, C, G, and T nucleotides are added sequentially to allow for synthesis of complementary strand. Unattached nucleotides are washed off
Pyrophosphate sequencing step 2:
Light is emitted when the nucleotide attaches to template. The sequence of light signals allows determination of DNA template sequence.
ATPs role in Pyrophosphate sequencing
ATP acts as fuel for luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP.
How is light captured in Pyrophosphate sequencing
Light produced in the luciferase-catalyzed reaction is detected by a camera/scanner
