Molecular Medicine

Question 1

molecular medicine

definition

Answer 1

application of molecular bio + biochem + genetics to diagnosis and treatment of disease

Question 2

common cause of breast/ovarian cancer

Answer 2

BRCA1/2 mutations
breast lifetime risk w/mutation= 45-85%
ovarian lifetime risk w/mutation= 39-46% (1) or 10-27% (2)

Question 3

PCR

Answer 3

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

Question 4

whole genome sequence

Answer 4

shows all SNPs, all mutations

more affordable and widely used, but remember 99% genome not expressed

Question 5

exome

Answer 5

aka exon portion, the 1% of the genome that is actually expressed

whole exome sequencing

Question 6

allele specific arrays

Answer 6

oligonucleotides bind just to WT or mutant sequence then fragment and label DNA

detect label by hybridization to mutant ASO indicates presence of mutation

Question 7

DNA sequencing

Answer 7

determine nucleotide sequencing

1. di-deoxy chain termination method - classic but only 1000 bp/reaction
2. next generation sequencing - miniaturization of reactions, larger sequence length but challenging to reconstruct

Question 8

SNP typing

Answer 8

only bits of genome that are known to vary

Question 9

DNA methylation sequencing

Answer 9

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

Question 10

FISH

Answer 10

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)

Question 11

cancer-MGPT

Answer 11

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

Question 12

prenatal diagnosis techniques

Answer 12

1. chromosome analysis i.e karyotype/FISH (detects numerical and structural aberrations)
2. exome sequencing (when karyotype is normal, single gene disorders)
3. cell free fetal (maternal)

turnaround time critical for reprod decisions and intrauterine intervention

Question 13

pharmacogenomics

Answer 13

develop an indiv drug treatment plan but response hard to predict with accuracy

i.e Warfarin (oral anticoagulant)

Question 14

Warfarin

Answer 14

inhibits vitamin K epoxide reductase (VKORC1) so polymorphism affects sensitivity

metabolized by P450 enzyme (CYP2C9) so polymorphism affects sensitivity

Question 15

considerations before testing

Answer 15

1. what to do with info aka will it help prevent disease or worry pt
2. who will have access- HIPAA, GINA (genetic discrim protection in employment and health insurance)
3. incidental findings
4. ethics

Question 16

qRT-PCR

Answer 16

for RNA analysis, normal PCR not work
-reverse transcriptase (RNA > cDNA)

shows amount of a few select RNA molecules in sample

Question 17

BRCA1 185delAG analysis

Answer 17

RNA missing 2 nucleotides
-transcript still presesnt
-not truncated transcript from stop codon

Question 18

protein analysis

Answer 18

use antibodies bc bind to antigens/proteins with high specificity

label/trace/precipitate antibodies

Question 19

monoclonal antibody production

Answer 19

1. challenge an animal w/ antigen aka inject into a mouse
2. isolate B cells (from spleen)
3. fuse B cells with immortal myeloma cells (cancer cells)
4. sep hybridoma cells
5. select clone that produces best antibody

Question 20

ELISA

enzyme linked immuno sorbent assay

Answer 20

assay highly quantitative for proteins, not size or localization

sandwich ELISA for id antigens OR
indirect ELISA for id antibodies

Question 21

blotting technique

Answer 21

DNA (southern) or RNA (north) or proteins (western)
-shows size well and quantity (not as well)

1. sep molecules in a gel
2. transfer blot onto membrane
3. detect molecule of interest

Question 22

western blotting

Answer 22

shows if protein is properly glycosylated (makes protein bigger)
or protein is cleaved

Question 23

mass spectroscopy

small molecules/metabolites

Answer 23

small moles ionized and sep by size

detector shows extremely accurate molecular weight so molecules id by weight

Question 24

molecular therapies

Answer 24

either target genes (replace defective or insert new) or proteins (antibodies destroy malignant cells)

Question 25

gene therapy | goal

Answer 25

aim to insert a functional copy of defective gene into genome thru viruses (DNA) inc specificity with CRISPR/cas9 guide RNA

Question 26

gene therapy delivery system

Answer 26

must be efficient to reach as many cells as possible but safely -cannot trigger fatal immune respone or transform into cancer cells

Question 27

ADA deficiency

Answer 27

adenosine deaminase deficiency = xs cellular dATP > inhibit DNA syn = SCID recessive disorder so ideal for gene therapy bc will dissapear if heterozygous state

Question 28

ex vivo gene therapy

Answer 28

1. isolate T cells from pt 2. transfect w/ virus carrying normal gene (ex. ADA) 3. select transfected T cells 4. infuse pt 5. test pt immune response

Question 29

CART | chimeric antigen receptor T cells

Answer 29

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

Question 30

cancer immunotherapy | naked antibodies

Answer 30

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

Question 31

cancer immunotherapies | conjugated antibodies

Answer 31

can be radiolabelled or chemo-labelled to deliver cytotoxic drug into center of tumor

Question 32

karyotype analysis

Answer 32

only on nucleated dividing cells from amniotic fluid or chorionic villi culture and stop in metaphase for test (2 weeks) time consuming and expensive

Question 33

aneploid pregnancies

Answer 33

45, X + trisomy 18 + 13 usually lost XXX/XXY/XYY + trisomy 21 chance to come to term (greater than 60%)

Question 34

complications of down syndrome

Answer 34

1. congenital heart defect 2. bowel obstruction 3. hearing loss 4. spinal problems 5. eye abnormal 6. leukemia and polycythemia 7. cognitive impairment 8. risk for alzheimer

Question 35

causes of down syndrome

Answer 35

1. free trisomy 21 (nondisjunction, low recurrence, majority) 2. translocation (recombination 14/21, high recurrence, aka familial DS) 3. mosaicism (nondisjuction, low recurrence, unclear outcome)

Question 36

edwards syndrome

Answer 36

full trisomy 18 characteristic ultrasound findings serious physical/mental disabilities -heart defects can be surgery | second most common trisomy

Question 37

gynecomastia

Answer 37

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

Question 38

turner syndrome

Answer 38

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)

Question 39

viable aneuploides

Answer 39

trisomy 21- down syndrome, most common trisomy 18- edwards syndrome, rare but 10% survive 45,X - females, turners syndrome 47, XXY- male, klinefelter syndrome

Question 40

fragile X syndrome

Answer 40

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)