Unit 4 Flashcards

Question

Q: In a pharmacogenetic test for CYP2D6 reported using the pharmacogenetic start nomenclature, what does the number 4 represent if the varient identified was CYP2D6\*4C

Answer 1

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Predict development of dieases
Better understand disease mechanisms
Improve successful rate of drug development
Facilitate precision medicine

Answer 2

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Menelian:

mostly early onset
high penetrance
known pattern of inheritance (ex: autosomal dominant)
low enviornmental input

Complex:

mostly late onset
low penetrance
some familial, but mostly sporadic
moderate/high enviornmental input

Answer 3

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Genetic and environmental controls that would not be possible in humans
Ability to get tissues that you cannot collect in humans
Genetic and phenotypic similarity of humans
Preclinical studies

Answer 4

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Strains of interest selected
Mouse crosses performed
Genetic regions controlling traits/disease identified
Underlying gene identified

Types of crosses: incross, outcross, backcross, intercross (image)

Answer 5

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Inbred strains

Brother-sister mating or >20 generations.
- Result: more than 99.9% of genome identical
Classical inbred strains derived from pet mice
Wild-derived inbred strains derived from wild mice

Outbred stocks

Genetically undefined
Useful when precise genotype isn’t important
Useful as pseudopregnant or foster mothers

Derivatives on inbred strains

Coisogenic strains
- If mutation of gene occurs within an inbred strain, mutant animal differs at only one locus from non-mutant animals of same strain
- Mutations can be spontaneous, induced, or generated through genetic manipulation approaches
- KO mice genearted using ZFN, TALEN, or CRISPR
Congenic strains
- Donor genomic region of interest –> recipient strain
- Can be used to confirm the genetic region affecting the phenotype
- Once a congenic is created, have model for physiological testing
- KO mice generated using backcrossing to transfer KO allele to a different inbred strain
Consomic strains
- Donor chromosome –> recipient strain
- Used to quickly identify chromosome that plays a role in a phenotype
Conplastic strains
- Donor mitochondrial genome –> recipient strain
Recombinant inbred animals
- Conventional recombinant inbred (RI) strains formed from initial cross between two different strains followed by an F1 intercross and 20 generations of brother-sister mating
- Contain blocks of genome from each parental strain on each chromosome
Collaborative cross strains
- “next generation” recombinant inbred strains
- Mixes up genome from eight inbred strains and then create 1,000 inbred lines
- Experimental model system that more accurately reflects the genetic structure of human populations
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Answer 6

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First need to select strain of interest and then perofm mouse crosses if needed (see image)

Using genetic markers for genetic mapping

Identify marker (SNP) correlated with phenotype
Gives region causal gene is likely located

LOD score plot

Test to compare the likelihood that two loci are linked vs the likelihood that the two loci are unliked
Is the genetic marker correlated with the causal region?

To confirm: make congenic strain. Move implicated region to recipient strain.

Then make subcongenic strains to further define the region. If this is a no go, there could be two genes within congenic strains.

Using consomic strains

Genetic mapping using recombinant inbred strains

Phenotype can be repeatedly analyzed using mice with the same genetic background
Genotype needs to be analyzed only once for each line
Can be backcrossed to the parental strains to further define the region of interest

Genetic mapping using CC mice

Over 38M genetic polymorphisms identified among the 8 CC founder strains
Marker based statistics: genotype marker
8-allele models: consider what founder strain it came from

Answer 7

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Mouse genome informatics

used to identify genes within a region
can see info on gene - does it relate to phenotype of interest?

Answer 8

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Genetic polymorphisms can affect a genes function by introdcuing an amino acid change, splicing site, or premature stop codon. Can also affect expression level by altering promoter, enchnacer, non-coding RNA, mRNA/protein stability, or introducing large deletion.

Sequencing performed by sanger sequencing or next-generation sequencing (NGS)

When you identify a polymorphism, look for variants within regulatory regions/splice sites/evolutionarily conserved regions, deletions/insertions, aa changes that change protein function.

Gene Expression Profiling: measuring difference between expressed genes between cells or tissues. Can identify pathways/genes involved in disease. Identification of potential candidate disease genes.

Determine transcript abundance

quantitative RT-PCR
gene expression arrays
RNA sequencing - whole transcriptome analysis

Expression quantitative trait loci (eQTL) analysis

the discovery of genetic polymorphisms that explain variations in gene expression
consider the expression of a gene as a trait (a phenotype)
cis-eQTL - position of the eQTL maps near the physical location of the causal gene (about 1 Mb upstream/downstream)
trans-eQTL - position of the eQTL does not map near the physical location of the causal gene (even on a different chromosome!)

Answer 9

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Rodent knock-out and knock-in models

Embryonic stem cells
Zinc-finger nucleases, TALENS, CRISPR/Cas9

In vitro assays

case to case
If enzyme, can potentially make recombinant protein to change enzyme back and see if that affects function

Answer 10

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Q1

Screen inbred strains for high BP and low BP
Outcross high BP x low BP
Backcross F1 with low BP inbred strain
Screen offspring for high BP phenotype
Use QTL analysis to identify which SNPs correlate with high BP - determined by LOD score
To confirm: make congenic strains by moving region of interested into recipient strain.

Q2:

Make subcongenic strains
Cross congenic strain x low BP inbred strain. Backcross F1 with low BP inbred strain (see image)
Screen BP

Q3: 1) Use bioinformatics to look at sequence differences between high BP inbred strain and low BP inbred strain in the 30 genes. With the genes containing sequence variability, use ‘mice genome informatics’ to search described functions. Begin by making knock-outs of genes described to have functions related to BP.

2) Look for up/down regulated genes within region of interest by using qRT-PCR or gene expression arrays as disregulation can contribute to disease.

Answer 11

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An eQTL is a locus that contributes to genetic variance of a gene expression phenotype. In eQTL analysis, gene expression is associated with genetic variation markers.

cis and trans eQTL refer to proximity of the marker to the gene they regulate. cis eQTLs are 1 Mb upstream/downstream of the gene. trans eQTLs are not located near the gene - they can even be on other chromosomes!

Gene expression varies cell-cell. Need to make sure variance in gene expression is corrleated with the disease and not an artificat of cell-type.

Expression data from eQTL analysis can be used to identify genes and pathways whose disregulation contributes to disease.

Answer 12

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Linkage and association studies

Linkage studies: correlate the inheritance of genotype with phenotype
Association studies: correlate the presence of genotype with phenotype
- Biased approach: test candidate genes based on what is known of the disease and the hypotheses of the mechanism
- Unbiased approach: scan whole genome for evidence of loci that cause the disease phenotype

Linkage disequilibrium (LD): nearby loci are in LD when recombination events occur between them very infrequently.

D’: inversely related to the fraction of chromosomes that have had historical recombination between them
r^2: correlation between two markers across chromosomes within a population
Haplotype: set of DNA variations or polymorphisms that tend to be inherieted together
DNase I hypersensitivity peak: suggests reguatory region

Genome wide association studies

Markers scanned across genomes of many people to find genetic variations associated with a particular disease
Uncovers risk factors in an unbiased way - finds novel biological mechanisms and pathways
4 parts:
- 1) select large population of individuals with disease and suitable comparison group
- 2) DNA isolation, genotyping, and data review to ensure high genotyping quality
- 3) Statistical tests for associations between the SNPs passing quality thresholds and the disease/trait
- 4) Replication of identified associations in an independent population sample or examination of functional implications experimentally

Answer 13

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If now evolution is occuring, then an eqilibrium of allele frequencies will remain in effect in each succeeding generation of sexually reproducing individuals

p = frequency of allele A1 in the population

q = frequency of allele A2 in the population

p²= percentage of A1A1 homozygous indiviaul

q² = percentage of A2A2 homozygous individuals

2pq = percentage of A1A2 heterozygous individual

p+q=1

p²+2pq+q²= 1

Answer 14

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Fine mapping to identify causal genes

Genotype-phenotype analysis

Testing candidate genes in animal models and humans cells

Answer 15

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Prediction

Drug target

Answer 16

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Identifies SNPs associated with disease by genotyhping many SNPs of individuals with the disease (case) and without the disease (control).

4 parts:

Selection of individuals with and without the disease
Genotyping of many, many SNPs
Statistics to determine which SNPs are associated with the disease
Replication of identified associated SNPs in independent populations and/or functional assays of identified associated regions.

Answer 17

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Q1:

p² + 2pq + q²= 1

p + q = 1

AA = p²

Aa = 2pq

aa = q²

Q2: q² = 1/10,000 so q = 0.01

1-0.01 = 0.99 so p = 0.99

frequency of carriers = 2pq so (2)(0.99)(0.01) = 0.0198

Answer 18

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Therapeutic drug monitoring (TDM) is the clinical practice of measuring specific drugs at designated intervals to maintain a constant concentration in a patient’s bloodstream, thereby optimizing individual dosage regimens. It is unnecessary to employ TDM for the majority of medications, and it is used mainly for monitoring drugs with narrow therapeutic ranges, drugs with marked pharmacokinetic variability, medications for which target concentrations are difficult to monitor, and drugs known to cause therapeutic and adverse effects.

Narrow therapeutic index (TD50/ED50)
- toxic dose/effective dose
Defined therapeutic range and toxic threshold
Concentration of drug in blood correlates with therapeutic/toxic effect
The dose and concentration of of the drug in blood correlate poorly
Series consequences if under-or-over dosed
Significant inter-individual variation
Knowledge of drug concentration in blood influences management

Identify commonoly monitored drugs

anti-epileptics
anti-fungals
immunosuppressants
antibiotics
psychiatric

Answer 19

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Pharmacokinetics: studies effect of the body on the drug

CLADME: compliance, liberation, absorption, distribution, metabolism, excretion

Pharmacogenetics: studies association of specific gene variants with changes in pharmacokinetics and/or pharmacodynamics

Describe factors that affect drug efficacy
- individual metabolism
- protein binding
Identify gene-drug pair candidates for pharmacogenetic monitoring
- Codeine (prodrug) –> morphine by CYP2D6
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