Molecular Medicine Flashcards
molecular medicine
definition
application of molecular bio + biochem + genetics to diagnosis and treatment of disease
common cause of breast/ovarian cancer
BRCA1/2 mutations
breast lifetime risk w/mutation= 45-85%
ovarian lifetime risk w/mutation= 39-46% (1) or 10-27% (2)
PCR
amplify DNA by sep template strands then bind oligonucleotides (primers) for polymerase to extend/amplify b/t primers
shows known polymorphisms in selected regions, single genes/locus
olig can be allele specific
whole genome sequence
shows all SNPs, all mutations
more affordable and widely used, but remember 99% genome not expressed
exome
aka exon portion, the 1% of the genome that is actually expressed
whole exome sequencing
allele specific arrays
oligonucleotides bind just to WT or mutant sequence then fragment and label DNA
detect label by hybridization to mutant ASO indicates presence of mutation
DNA sequencing
determine nucleotide sequencing
- di-deoxy chain termination method - classic but only 1000 bp/reaction
- next generation sequencing - miniaturization of reactions, larger sequence length but challenging to reconstruct
SNP typing
only bits of genome that are known to vary
DNA methylation sequencing
bisulfite sequencing (coverts C to U) then PCR converts (U to T)
C bases that were converted to T were NOT methylated in og sample
tissue specific
FISH
hybridize labelled DNA probe to genomic DNA in interphase nucleus
-should be 2 fluorescent sites for autosomal loci
-3 loci = region is present in 3 copies (trisomy)
-1 loci = one copy (monosomy)
cancer-MGPT
MGPT (multigene panel testing) to determine seq of known oncogenes and tumor suppressors
classifications: pathogenic, likely patho, uncertain significance, likely benign, benign
can predict risk/prognosis/suggest treatment
prenatal diagnosis techniques
- chromosome analysis i.e karyotype/FISH (detects numerical and structural aberrations)
- exome sequencing (when karyotype is normal, single gene disorders)
- cell free fetal (maternal)
turnaround time critical for reprod decisions and intrauterine intervention
pharmacogenomics
develop an indiv drug treatment plan but response hard to predict with accuracy
i.e Warfarin (oral anticoagulant)
Warfarin
inhibits vitamin K epoxide reductase (VKORC1) so polymorphism affects sensitivity
metabolized by P450 enzyme (CYP2C9) so polymorphism affects sensitivity
considerations before testing
- what to do with info aka will it help prevent disease or worry pt
- who will have access- HIPAA, GINA (genetic discrim protection in employment and health insurance)
- incidental findings
- ethics
qRT-PCR
for RNA analysis, normal PCR not work
-reverse transcriptase (RNA > cDNA)
shows amount of a few select RNA molecules in sample
BRCA1 185delAG analysis
RNA missing 2 nucleotides
-transcript still presesnt
-not truncated transcript from stop codon
protein analysis
use antibodies bc bind to antigens/proteins with high specificity
label/trace/precipitate antibodies
monoclonal antibody production
- challenge an animal w/ antigen aka inject into a mouse
- isolate B cells (from spleen)
- fuse B cells with immortal myeloma cells (cancer cells)
- sep hybridoma cells
- select clone that produces best antibody
ELISA
enzyme linked immuno sorbent assay
assay highly quantitative for proteins, not size or localization
sandwich ELISA for id antigens OR
indirect ELISA for id antibodies
blotting technique
DNA (southern) or RNA (north) or proteins (western)
-shows size well and quantity (not as well)
- sep molecules in a gel
- transfer blot onto membrane
- detect molecule of interest
western blotting
shows if protein is properly glycosylated (makes protein bigger)
or protein is cleaved
mass spectroscopy
small molecules/metabolites
small moles ionized and sep by size
detector shows extremely accurate molecular weight so molecules id by weight
molecular therapies
either target genes (replace defective or insert new) or proteins (antibodies destroy malignant cells)
gene therapy
goal
aim to insert a functional copy of defective gene into genome thru viruses (DNA)
inc specificity with CRISPR/cas9 guide RNA
gene therapy delivery system
must be efficient to reach as many cells as possible but safely
-cannot trigger fatal immune respone or transform into cancer cells
ADA deficiency
adenosine deaminase deficiency = xs cellular dATP > inhibit DNA syn = SCID
recessive disorder so ideal for gene therapy bc will dissapear if heterozygous state
ex vivo gene therapy
- isolate T cells from pt
- transfect w/ virus carrying normal gene (ex. ADA)
- select transfected T cells
- infuse pt
- test pt immune response
CART
chimeric antigen receptor T cells
genetically engineered T cells detect and destroy cancer cells
-T cells from pt > transfect w/ lentiviral vector = chimeric receptor for tumor cell antigen so CART cells recognize lymphoblasts
acute lymphoblastic leukemia treatment
cancer immunotherapy
naked antibodies
naked antibodies
1. bind to tumor cells and attract immune cells to destroy cancer
2. unblock immmune system
3. block receptors on cancer to shut off growth signals
cancer immunotherapies
conjugated antibodies
can be radiolabelled or chemo-labelled to deliver cytotoxic drug into center of tumor
karyotype analysis
only on nucleated dividing cells from amniotic fluid or chorionic villi
culture and stop in metaphase for test (2 weeks)
time consuming and expensive
aneploid pregnancies
45, X + trisomy 18 + 13 usually lost
XXX/XXY/XYY + trisomy 21 chance to come to term (greater than 60%)
complications of down syndrome
- congenital heart defect
- bowel obstruction
- hearing loss
- spinal problems
- eye abnormal
- leukemia and polycythemia
- cognitive impairment
- risk for alzheimer
causes of down syndrome
- free trisomy 21 (nondisjunction, low recurrence, majority)
- translocation (recombination 14/21, high recurrence, aka familial DS)
- mosaicism (nondisjuction, low recurrence, unclear outcome)
edwards syndrome
full trisomy 18
characteristic ultrasound findings
serious physical/mental disabilities
-heart defects can be surgery
second most common trisomy
gynecomastia
hx of behavior problems(societal, not genetic), normal IQ, learning disabilities
-microordchidism + tall (95th percentile) + dev breast + high pitched voice
-infertile (azoospermia)
47, XXY (extra x/barr body, active Y so male)
klinefelter syndrome
turner syndrome
infant: low posterior hairline + webbed neck + lymphedema + cardiac murmur
child: short, shield chest (SHOX), ovarian failure, horseshoe kidney, coarctation of aorta
45, X (affects females, high fetal loss from lymphatetic malformation)
viable aneuploides
trisomy 21- down syndrome, most common
trisomy 18- edwards syndrome, rare but 10% survive
45,X - females, turners syndrome
47, XXY- male, klinefelter syndrome
fragile X syndrome
CGG triplet expansion in 5’UTR of FMR1 gene (beyond 200 units = silence of FMR1 locus via methylation)
evident only in folate deficient media, use PCR or southern blotting
2nd most common cause of ID (down syn is 1st)
