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MedGen Ex3 - 13,17,18

Terms in this set (67)

CYSTIC FIBROSIS!! at diff levels

1) mutant gene - mutant CFTR gene (receptor allowing transport of Cl ions), how do you fix the DNA seq itself (make it wildtype seq)
modify somatic genome
A- transplantation - bone marrow where gene is correct in cell being donated - not CF bc receptor needs to be active in basal ganglia
B- gene therapy - CRISPR

- pharmacological modulation of gene expression/transcription - inc HbF in sickle cell disease - inc expression might not help depending on protein structure/function (determined by location/type of mutation in each person) - not fixing mutant gene just changing exp of it, not for CF bc a lot of allelic heterogeneity (multi-allelic) - not truly homos, compound heterozygotes in autosomal rec disorder, diff alleles make diff proteins (which all look a little different, LOF vs potentiator and corrector)

2) mutant mRNA - CF mutant CFTR transcribed mRNA
- RNA interference to degrade mutant mRNA - too much of something, RNAi degrades and breaks it down to more normal levels not good example, bc there is no particular allele involved - not CF, bc no particular allele where the problem is "too much of something", all broken CTFR genes

3) mutant protein - CF integral transmembrane protein with Cl channel,
- protein replacement - skipping of the stop codon, just give them a good protein
- enhancement of residual function - depending on allele, some may have residual function, just have to get them to the location where they can carry it out
CF: if stop codon causes broken CTFR gene, can be skipped with exon skipping, cant just give them CFTR bc has to be trafficed to certain place in membrane (correctors)

4) metabolic or biochemical dysfunction - e/chem gradient is dec
- disease-specific compensation - 1) dietary - high salt diet specific to what alleles they have (residual function or not), 2) pharmacologic - infused with saline solution
CF: diet (high salt) only with alleles of CTFR gene with residual function, help Cl ions stay in - pharma (saline solution in IV)

5) clinical phenotype - cl seen in high levels in sweat
- medical intervention - blood transfusion
- surgical intervention - lung transplants, vest for percussion treatment
CF: med: lung transplant

6) the family
- genetic counseling - partner to be screened
- carrier screening - for having kids
- presymptomatic diagnosis - no presymptomatic testing
CF: carrier for CF (auto rec)
presently, the most successful disease-specific approach to the treatment of genetic disease is directed at the metabolic abnormality in inborn errors of metabolism. The principal strategies used to manipulate metabolism in the treatment of this group of diseases are listed in Table 13-2. The necessity for patients with pharmaco- genetic diseases, such as glucose-6-phosphate dehydro- genase deficiency, to avoid certain drugs and chemicals is described in Chapter 18.

lots of enzymopathies

- avoidance - if a particular drug or env component (chem) makes disease worse, you should avoid - could be anything (might be dietary restriction, so overlap)

- dietary restriction - avoiding food specifically, like PKU, and how low phenylalanine diet can help, most common - PKU dec phenylalanine

- replacement - if you have something missing can we give it to you? (cofactor, protein) low levels of X treated with drugs that have X in it

- diversion - diverts the pathway by using alternative metabolic pathway, no specific drug or anything, deficiency in enzyme that plays an early role in the cycle, buildup of what comes before in the cycle, this gets diverted/forced to another pathway, doesn't cause negative effects, fig 13.4 + .5

- enzyme inhibition - receptors allow some chemical to come in from plasma, feedback mechanisms that inc/dec enzyme synthesis of that compound once it comes in without a drug (that one fig) - NO DRUG NEEDED (FH) - block de novo synthesis: bring more from plasma

- receptor antagonism - if problem is with receptor (wrongly activated), add drug that acts against it to stop it - connective tissue disorder (not w/CF)

- depletion - if harmful product accumulates, can directly remove them - FH homozygote (no good receptors, both genes mutated = no drugs work) can have plasma taken out and cleared and new back in, cholesterol is cleared from plasma and then put back in

(middle is diversion: drugs can divert it so it is excreted in feces, inc bile acid synthesis and dec intracellular cholesterol, and inc LDL conc, now can bring more in, statin and bile acid depletion have best effect, statins block HMG CoA, inc right side of the pathway, bringing more cholesterol into the cell through LDL receptor)
biochemical abnormalities of a diff metabolic diseases respond, sometimes dramatically, to the administration of large amounts of the vitamin cofactor of the enzyme impaired by the mutation (

enhancement of mutant protein function
- skipping drugs - (NOT EXON) drugs that can help translational (RNA) machinery skip over stop codon - drugs have to be targeted, you have to know genotype and specific alleles bc drug works only in those stop codons (still in DNA/mRNA, only at protein level - have to be able to target it (CFTR)

- correctors - protein (only) that needs to get to apical cell membrane, depending on folding, might not get to membrane, but if it does, can still do it's job - use a drug to help RECEPTOR protein (only) get through the ER to the CELL MEMBRANE


- potentiator/enhancer - protein has no problem getting to the membrane, but is just bad at its job, inc protein function at cell membrane, proteins have to be able of getting there without help - Orkambi???: lumacaftor and ivacaftor, helps with heterozygotes needing corrector and potentiator

- vitamin cofactors - depending on protein, mutation is in site where protein usually binds with cofactor,(partial or full response - so statistically, more chance) - sometimes enzyme is not active until it binds to cofactor, so creating more cofactor = inc cofactor for protein to bind to, only if mutation is in cofactor-protein bidning site to be active, so statistically, more chance of being active = inc residual activity of mutant enzyme to give partial

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protein augmentation - ensuring individual has more of protein in the cells
· replace a mutant extracellular protein with a functional one - "just give them the EC good one" - rarely works

· replace a mutant intracellular protein - have to ask: does it have to be targeted to the tissue? can it get in the cell? does it have to be at a certain place in the cell? (mutation in a gene, helps stabilizes individuals while waiting for bone replacement therapy - more successful) drug put in EC but may be targeted for IC not going directly into the cell, cell will take it up

· replace an intracellular protein through cell targeting - going INSIDE the cell specifically (targeted)))
transplantation can be considered a form of gene transfer therapy bc transplanted cells retain donor genotype and lead to modifications of somatic genome

transplantation: marrow and stem cells from blood, particularly effective for blood disorders
- 10% mass = bone marrow transplant successful in diseases where swapping 10% of cells is enough to make a difference (help with symptoms of lysosomal storage disease)
- SCD and B-thalassemia

indicators: to put WT copies of mutant gene into pt, or for cell replacement

stem cell: proliferate to form cell types of tissues, and can self renew (make more), 3 types:= hematopietic (blood system) used for non-storage (SCID, B-thal and SCD), and lysosomal storage disorders (hurler?)

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- 10% of body mass is bone marrow
- quantitative impact of enzymes transferred from bone marrow transplantation = corrects or reduces the visceral abnormalities of many storage diseases
(if there is a disease that marrow can make a difference in, match is relatively easy way to modify the somatic genome)
- transplanted cells maintain their genome

- only if that 10% change is enough - examples are hurler syndrome - enzyme defect results in sugar buildup in lysosome, have found that marrow transplant helps with symptoms - ALSO sickle cell disease and thalassemia - but very dependent on type of disease/genome involved

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- 1st, cells or organs transplanted to introduce wild-type copies of a gene into a patient with mutations in that gene
- 2nd. cell replacement, to compensate for an organ damaged by genetic disease
- DMD x-linked REC, makes dystrophin which stabilizes sacrolemma in smooth, skeletal, cardiac muscle
- DMD mut: large deletions (ooo), duplications, small del/ins/nucleo changes (CpG, sperm)
- dynstophin res fx = less severe pheno, but all 95% with cardiac compromise, 50% chronic heart failure, low IQ
- fem age of onset depends on X-inactivation: if x chr with allele active: signs of DMD, other: none/few - still have cardiac abnormalities if carrier fem
- diagnosis: family history glucocorticoid, therapy to slow dis, and maintain mobility, weight, pulmonary/cardiac function, scoliosis, gene transfer???

- carrier mom = 50% risk for son w/DMD, 50% of girl inheriting DMD allele but not developing DMD bc random x-inactivation, if not a carrier mom = boy with germline mosaicism

bc dystrophin hass so many exons, carrier screening is hard

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- X LINKED REC - allelic heterogeneity, manifestation, phenotype, high freq of new mutations
- muscle weakness, calf pseudohypertrophy, mild int dis, inc serum creatine kinase levels (gait, cardiac issues) in 1/3500 boys
- dystrophin of DMD expressed in smooth, skeletal and heart muscle, and part of sarcolemma stability proteins - manifesting in newborn period - make dystrophin null = severe, res function - not
- caused by LARGE deletions (oogenesis), duplications, small deletions, insertions, nucleotide changes (spermgen)
- early in males, 95% males have cardiac compromise and 50% chronic heart failure BUT late in females
- therapy: slow dis, maintain mobility, correct scoliosis, weight, glucocorticoid therapy (slows DMD) and inv. gene therapy
- 1/3 of moms with affected son arent carriers, hard to find carrier state bc large number of EXONS in dystrophin gene and germline mosaicisim


carrier mom: 50% risk of DMD in son, 50% risk of DMD mutation inheritance (but low risk of getting DMD bc random X-inactivation)

not carrier: still give to mom bc of germline moasaicism
in prok: CRISPR Cas-9 is natural for bacteriophage
1) locus transcribed w/pre crRNA comp to invading seq with....
2) guide chimera of crRNA (breaks viral DNA) and tracrRNA (conn/stab CRISP-Cas9) complex
3) Cas 9 uses helicase (unwind) and nuclease (cut)

euk: not natural, so you have to give them crRNA and tracrRNA as 1 part instead of 2, or wont work

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- crispr cas 9 makes cut naturally in bacteria (prok)
- new part: developing it for use in eukaryotic cells, all based on repair mechanisms (NHEJ or HDR, homology directed prepare

- CRISPR Cas-9 makes cut, not new - new part is developing it for use in eukaryotic mechanisms, based on cell mech

- CRISPR = clustered regularly interspaced short palindromic repeats

·- CRISPR = naturally found in bacteria (prokaryotes) and it is used for bacteriophage, artificial in eukaryotes

- when the locus gets transcribed: pre-crisper RNA (has lots of RNA pieces), when you cut them out, will be attached to a piece complementary to guide RNA (chimera)

- 2 enzymatic activities of Cas proteins = helicase: unwind DNA, nucleases: cut DNA
· native bacterial system crRNA = complementary to one of the invading viral sequences, breaks viral DNA

- native bacterial system tracrRNA = seq that connects/link/adaptor between CRISPR RNA and Cas-9 complex w/RNA in it - holds CRISPR RNA in place, diff protein units to bind in, comes from another sorts

- artificial eukaryotic CRISPR-Cas9 technology tracrRNA-crRNA chimera = guide RNA, not 2 separate parts that come together in cell like in prokaryotes (guide RNA: tracr RNA + crisper RNA), but made as one and added as a unit in eukaryotes that is specific to seq needed to be cut, and also with Cas-9 binding sequences

· *** euk: get both guide RNA and Cas-9 as 1 part
* X-linked DOM: 99% caused by 5'UTR CGG ~200 repeat expansion mutation in FMR1 gene (leading to hypermethylation that causes loss of FMRP chaperone protein exp)

* full CGG exp rep mutations of FMRI come from mom w/mutant premat allele (not dad, he shortens premutations) - mat premat tran bias - affect more kids from gen to later gen (anticipation)

* premature progression to full mutation inc w premutation length , but rare, only in haplotypes (haplo predisposition - lack of AGG which inh expansion) - all males and 50% of fem with full mutation = FXS

- full mutation is unstable so mosaicism exists (some cells w/prem and some w/full length # repeats) and methylation (some have cells w/w/o methylation of CGG exp repeat

* males w/mosaicism and methylation affected but have higher mental function than those w/full mutation in each cell + hypermethylation in every cell WHILE fem phenotype dependent on X inactivation

* fem w/premut but w/o full have ovarian failure risk and have carrier risk of having 25% affected girl, 50% affected boy depending on premut size

* males w/premut but w/o full have FXTAS risk

FXS IQ dis moderate in males, mild in fem + behavioral abn, males have more distinct ft - normal life span - FETAL CELL DNA SCREENING from cvs or amnio

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x linked DOM - triple repeat expansion, somatic mosaicism, sex-specific anticipation, DNA methylation, haplotype effect
- FMRP of FMR1 gene (neurons) chaperones mRNAs to translational machinery

risk that a fem with premutation will have affected child determine by size of premutation, sex, family history - 50% if males, 25% if fem

(FETAL DNA TEST USED - mom - prenatal testing)
- hypothesized that FRMP is a mRNA carrier chaperone protein that transports mRNA from the nucleus to the cytoplasm for translation? (not a DNA-binding chaperone)

- hypermethylation in the FRMP is responsible for FX

- mother with a premutation or a father with a premutation would be more likely to pass on a full mutation - even though dad goes through more rounds of replication, maternal transmission bias for repeat expansions in fragile x syndtrome

- FXS: anticipation disorder (larger it gets, stronger the phenotype), trinucleotide repeat expansion disorder = earlier age of onset, expanding CTG = more methylation - larger expansion of CTG, : more methylation, stronger the phenotype or earlier age of onset

- more likely to affect males vs females (bc 1 x vs 2)

- which best describes the intellectual disability associated with FXS? moderate in males and milder in females

- severity of phenotype depends on:
* repeat length mosaicism (how many cells have expansion repeat)
* skewed x inactivation in females (female who is carrier has more severe phenotype is good X got inactivated)
* repeat methylation mosaicism (how many cytosines methylated in diff cells)

- a potential treatment for FXS is the use of small molecules that inhibit DNA methyltransferases (inh methylation of cytosines) - which of the following has been seen for this treatment - small molecules only work for a few days

- in the article, CRISPR-Cas9 was used to: delete the CGG repeats in the 5' UTR

- which of the following applies to the article's CRISPR-Cas9 strategy - NHEJ (not HDR, homology directed repair)

- in the article, how many cuts did they want the CRISPR-Cas9 system to make in the 5' UTR: 2 (NHEJ)
cas 9 makes DS break, eukaryote uses NHEJ to put them back together at risk of adding insertions and deletions (bc most DNA is noncoding, this isnt an issue)

NHEJ used to knockout gene for studying mutant genes, treats diseases caused by a toxic protein

CRISPR - DNA seq, RNAi - mRNA prevention of translation - CF: HDR (homology directed repair) where donor DNA is template for fixing it - double crossover event

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After the Cas9 makes double-stranded cuts in the DNA, a eukaryotic cell will try to correct the break— the system the cell uses is called non-homologous end-joining (NHEJ). This is a DNA repair system that puts broken DNA molecules back together again. The problem is it is not perfect—as a part of that process, oftentimes insertions and deletions (indels) occur within the DNA. Why would such a DNA repair system be evolutionarily selected for? Well, considering that most eukaryotic DNA is noncoding, the majority of the time, it is not a problem for indels to be created--the main point of NHEJ is to get the chromosome back together again.

But how could NHEJ be beneficial in CRISPR technology? Isn't the end goal to "fix" a gene, not mutate it? Well, originally this technology was developed as a means to knockout a gene in a eukaryotic organism so the effects of that knockout could be studied. For example, if we want to know what gene X does in mice, we could design a gRNA that targets gene X, couple that with Cas9 and insert into the mouse cells. This would mutate gene X because NHEJ will create indels in gene X.

Another way NHEJ could be beneficial with CRISPR is to treat diseases that are caused by a toxic protein (think about a heterozygote for a dominant disorder). Using CRISPR technology we could create a gRNA for the gene that encodes the toxic protein and knockout the toxic protein expression. You may be seeing some similarities to RNAi in this strategy, and you would be correct. The difference is CRISPR is changing the DNA sequence but RNAi works at the mRNA level by preventing its translation. Two different approaches that accomplish a similar goal-- but why would CRISPR be preferred over RNAi (think about this)?

So why all the hype about CRISPR? You have probably heard it can cure genetic disease, but can it only work for diseases that produce a toxic protein? Here is the super cool part... let's consider cystic fibrosis. If we put gRNA (specific to the CFTR gene mutation), Cas9, and a small piece of DNA with the correct nucleotides for the CFTR gene (often called "donor DNA") into the cell, the cell does not use NHEJ to correct the break that Cas9 creates. It does something called homology-directed repair (HDR), in which it uses the donor DNA as a template to repair the DNA, thus incorporating the correct sequence for the CFTR gene. WOW!!! This occurs via a double-crossover event-- if you are confused on how the crossovers work with HDR, look it up on YouTube-- there are some cool videos.
prenatal diagnosis: testing of fetus w/known inc risk for a genetic disorder to determine if the fetus is/isn't affected, where increased risk is determined due to previous afflicted children, family history, positive prenatal carrier test, or prenatal screening test
- known elevated risk
- usually invasive testing (CVS and amnio)
- "yes" or "no" answer
- determine if the fetus is/isn't affected

goal: inform abt risk for birth defects or genetic disorders and give them choices on how to manage it

prenatal screening: looks in pregnancies not known to be at an increased risk for defect/disorder to find defects like chromosomal aneuploidies, neural tube defects, and other structural anomalies - is also typically noninvasive
- no known elevated risk
- noninvasive (maternal blood sample, MRI and ultra)
- not a "yes" or "no" answer
- identifies pregnancies that should have prenatal diagnosis

goal: find that pregnancies that should have prenatal diagnostic testing

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prenatal diagnosis: testing of a fetus known to be at increased risk for a genetic disorder to determine if the fetus is/isn't affected, where increased risk is determined due to previous afflicted children, family history, positive prenatal carrier test, or prenatal screening test - Y/N answer (amniocentesis)

- prenatal screening test - looks in pregnancies not known to be at an increased risk for defect/disorder to find defects like chromosomal aneuploidies, neural tube defects, and other structural anomalies - is also typically noninvasive - inc risk + probability, not invasive
superfast is toxic with prodrug
slow is toxic with toxic drug

1) if drug is prodrug: drug PRODUCT is toxic:

· normal metabolizers: normal CYP genes = enzyme good, normal speed conversion of codeine to morphine

GOOD bc prod is toxic but loss of therapy · slow metabolizers: mutant CYP = codeine will be broken down slowly, so more codeine = not a toxicity issue, no overdose issue but no pain relief

BAD bc prod is toxic · ultrafast metabolizers: multiple copies of enzyme = fast conversion of codeine to morphine, hasn't been excreted so you have to be careful, could OD very quickly - dangerous so have to be careful

2) drug is not prodrug, product is not important - showing level of active (toxic drug): - drug that's given is toxic:

· normal metabolizer: metabolized, and excreted consistently, trying to keep levels of drug HIGH

BAD bc reactant is toxic · slow metabolizer: scary bc could be toxic at high levels, cant just keep getting it

GOOD bc reactant is toxic but loss of therapy · ultrafast metabolizer: so fast that you need to constantly add to even keep it in a range, might have trouble having therapeutic effects

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combinations of reduced, absent, or inc activity alleles produce quantitative differences in metabolizing activity, resulting in three main phenotypes: normal (also called "extensive") metabolizers, poor metabolizers, and ultrafast metabolizers (Fig. 18-3).

serum drug levels after repeated doses of a drug (arrows) in three individuals with different phenotypic profiles for drug metabolism.

A, Poor metabolizer accumulates drug to toxic levels (or have poor activation that leads to inefficient therapy) LOF - reduced alleles?

B, Normal (extensive) metabolizer reaches steady-state levels within the therapeutic range. - absent alleles?

C, Ultrafast metabolizer fails to maintain serum levels within the therapeutic range. - inc activity alleles
1. Watch this video:
https://www.youtube.com/watch?v=MnYppmstxIs (Links to an external site.)
2. What does CRISPR stand for?
3. CRISPR is naturally found in ________________________________, and it is used for ________________________________________________. The video illustrated how this works, but here is another figure as well:



4. What 2 enzymatic activities do Cas proteins have?
5. In the native bacterial system, what is crRNA?
6. In the native bacterial system, what is tracrRNA?
7. In the CRISPR-Cas9 technology developed for eukaryotes, what serves as the tracrRNA-crRNA "chimera?"

After the Cas9 makes double-stranded cuts in the DNA, a eukaryotic cell will try to correct the break— the system the cell uses is called non-homologous end-joining (NHEJ). This is a DNA repair system that puts broken DNA molecules back together again. The problem is it is not perfect—as a part of that process, oftentimes insertions and deletions (indels) occur within the DNA. Why would such a DNA repair system be evolutionarily selected for? Well, considering that most eukaryotic DNA is noncoding, the majority of the time, it is not a problem for indels to be created--the main point of NHEJ is to get the chromosome back together again.
But how could NHEJ be beneficial in CRISPR technology? Isn't the end goal to "fix" a gene, not mutate it? Well, originally this technology was developed as a means to knockout a gene in a eukaryotic organism so the effects of that knockout could be studied. For example, if we want to know what gene X does in mice, we could design a gRNA that targets gene X, couple that with Cas9 and insert into the mouse cells. This would mutate gene X because NHEJ will create indels in gene X.
Another way NHEJ could be beneficial with CRISPR is to treat diseases that are caused by a toxic protein (think about a heterozygote for a dominant disorder). Using CRISPR technology we could create a gRNA for the gene that encodes the toxic protein and knockout the toxic protein expression. You may be seeing some similarities to RNAi in this strategy, and you would be correct. The difference is CRISPR is changing the DNA sequence but RNAi works at the mRNA level by preventing its translation. Two different approaches that accomplish a similar goal-- but why would CRISPR be preferred over RNAi (think about this)?
So why all the hype about CRISPR? You have probably heard it can cure genetic disease, but can it only work for diseases that produce a toxic protein? Here is the super cool part... let's consider cystic fibrosis. If we put gRNA (specific to the CFTR gene mutation), Cas9, and a small piece of DNA with the correct nucleotides for the CFTR gene (often called "donor DNA") into the cell, the cell does not use NHEJ to correct the break that Cas9 creates. It does something called homology-directed repair (HDR), in which it uses the donor DNA as a template to repair the DNA, thus incorporating the correct sequence for the CFTR gene. WOW!!! This occurs via a double-crossover event-- if you are confused on how the crossovers work with HDR, look it up on YouTube-- there are some cool videos.
The following figure illustrates the 2 pathways mentioned above: NHEJ and HDR.


So this sounds great-- let's cure every genetic disease, right? Well, it's not that simple. Here are the issues:
1) How do we ensure that every cell gets corrected? For some diseases, we could CRISPR cells in a particular tissue and that would be enough to cure the disease, but if the disease impacts numerous systems, that gets more complicated. We could screen embryos and CRISPR them-- but that becomes an ethical issue.
2) The technology has not been perfected yet. Oftentimes, there are off-target changes (changes in other regions of the DNA that are similar to the gRNA sequence).

I highly recommend Jennifer Doudna's book "A Crack in Creation" for your reading list down the road.