Quant Genetics Final

what could a SNP in the exon cause

• premature stop codon

• frameshifts

• missense stability

what would a SNP in the exon effect

mainly effect function of the gene product

• protein stability

• change of protein sequence

• protein interaction

• protein-dna interactions

what would mutation in the promoter region cause

promoter region is responsible for expression of the target gene

• effects by changing a motif sequence, chromatin accessibility, transcription factor binding

how would you test for the causal effects of SNP in the promoter region

• purify the normal and mutant proteins to test their functions in vitro

• express protein in cell lines or model organisms to test function in vivo

how would you test for the causal effects of SNP in the exon region

1. transcription factor motif analysis to see if specific motif is disrupted by snp

2. luciferase assay to determine whether snp affect transcription

3. atac seq and histone modification chip seq to determine whether chromatin accessibility is affected or not

what are the 3 genetic explanations for the missing heritability problem

1. infinitesimal hypothesis: common variants have small additive effect sizes BUT responsible for most of var in the pop. (strongly supported)

2. rare allele hypothesis: rare alleles have large effects

3. epistasis hypothesis: extensive epistasis hidden in heritability studies (not well supported)

rare variants can have large effect on disease. give an example

• GH1 deficiency

• has large effect on height

• but is rare → little effect on overall variance for height in human pop

we know that common variants have small additive effect but are responsible for most of the variance in a population + rare variants can have large effect but contribute little to variance.

explain this idea in terms of evolution

1. if mutation causes big harmful effect, natural selection will select against it (GH1 deficiency)

2. trait that is controlled by many variants (each with small effects), none individually is strong enough to trigger negative selection + preservation of genetic diversity

what is GCTA (Genome-wide Complex Trait Analysis)

• using a mixed linear model

• tool that combines the effects of ALL snps (not just the significant ones) to estimate h²

• can severely overestimate or underestimate h² because population structure due to admixture

population admixture def

• people from 2 dif ISOLATED places intermix

• making a new mixed population

how do false positive occur from population admixture

• failure to take mixed genetic background into account → false (+) in GWAS

• why? alleles that distinguish the 2 pops will appear to be associated with the trait

what is protective varaint

genetic change that reduces the risk of developing a disease.

what is a haplotype

• chunk of DNA variants (alleles or SNPs) where a set of variants are strongly linked

• why? located close to each other on the same chromosome.

explain linkage disequilibrium and the problem that arises from LD

• LD = physically close variants tend to be inherited together

• why? closeness = less recombination

• problem: some allele combinations are over represented → SNP might look associated with a trait but is really just “tagging along”

4 steps to find the responsible gene and mutation

• extend association study to tracing inheritance in families

• predict effect of mutation on gene func

  • stability? expression?

• verify effect on gene func

  • Express normal and mutant proteins in cell lines and test functions.

  • Luciferase assays to test the promoter activity.

  • ATAC-seq and ChIP-seq for chromatin accessibility

• verify effect on phenotype

  • gene knockout

what is cytological mapping and what do we use it for

• technique to locate structural abnormality (deletions, inversions, translocations) on chromosome that may lead to

• inactivate or inappropriately activate a gene

• specifically we want to look at chomosoal abnormalities

when would cytological mapping come in handy

1. rare disease traits with not enough pedigree

2. Highly deleterious dominant traits

   1. trait so harmful it does not get passed down aka the person dies

what are the two methods that we could use to help use identify mutations in the genome

1. cytological sequencing: sequencing of the entire genome

   1. structural abnormalities (deletions, inversions, translocations)

2. exon seq: sequencing of just the exons

   1. look for SNP in the protein coding region

what is exome sequencing and what do we use it for

much like cytological mapping we sequence only the exons and look for SNPs

• use for single gene disorders that are

• very rare disease traits with not enough pedigree

• highly deleterious dominant traits (deadly)

what are the 2 things that could result in chromosomal abnomalities

1. inversions and deletions

2. translocation (result from breaks on different chromosomes)

what are some limitations of exon sequencing

1. relevant mutations are not necessarily on exons or protein coding regions

2. not useful for complex traits (trait that is influenced by many factors)

what is a SNP

a single nucleotide is changed

what are CPG islands and why do they matter

• sequence of DNA with high CpG content

• CPG islands are located in promoters

• If CpG island becomes methylated by DNA

  Methyltransferase, it usually silences the gene downstream

what is gene imprinting? why do we care? what are examples of diseases associated with imprinting?

• 1 gene is methylated (silent), 1 is unmethylated (expressed)

• can either be the maternal or the paternal

• Prader-Willi syndrome: loss of paternally expressed genes

• Angelman syndrome: loss of maternally expressed genes

2 methods that control gene expression

1. histone modification

2. methylation

what does bisulfite seqeuncing do

tell you where the dna is methylated and not methylated

we talk about cis and trans acting regulation of gene expression. what does that mean

cis acting: promoters, enhancers, insulators

trans acting: transcription factors

what is a promoter

• dna seq upstream of a gene

• start site where RNA polymerase binds

• essential for gene expression

think of it as an on and off switch transcription ex (TATA box)

what is an enhancer

• DNA regions that can boost the transcription of a gene

• Enhancers bind transcription factors

• Usually upstream of the promoter

  but can be far upstream, downstream,

  or within a gene (in introns)

A volume knob that amplifies gene expression — but only works when the right transcription factors are present.

what is a transcription factor

• Proteins that bind to specific DNA sequences (in promoters or enhancers).

• helps to activate or suppress transcription

what are locus control regions

An LCR is a long-range enhancer that controls multiple genes in a gene cluster.

• think master regulator

what is chromatin

DNA wrapped around histone proteins

how do histones affect gene expression?

makes it hard for transcription factors and transcriptional machinery to access DNA…

what is epigenetics

study gene expression that do not involve changes to the DNA sequence itself ie methylation, Histone Modifications etc

chromatin states arise from DNA and chromatin modifications. what 2 things would result in open chromatin? condensed chromatin?

• condensed: dna methylation, histone deacetylation

• open: dna demethylation, histone acytylation

what does dna methylation mean? what is the function of DNA methylation? histone acetylation?

• dna methylation: attaching a methyl group onto a cpg group (found in cpg island)

• function: mechanism to control gene expression

• histone acetylation: attaching an acetyl group, also control DNA methylation

what is the difference between de novo methylation and methylation maintenance

• de novo methylation: establish a repressed state in response to developmental program or environmental factors 

• Maintenance methylation: maintains repressed state through somatic cell divisions

If methylation silences gene expression and it is heritable through cell division, how can new progeny develop?

Methylation status is controlled dynamically (erased during dev of germ cells then reset during subsequent differentiation of gametes, fertilization, and development.

what is the purpose of taking folic acid when women are pregnant?

folic acid increases methylation. deficiency in dna methylation is associated with neural tube defects.

when does imprinting occur

during germ cell differentiation

explain the parent of origin effects

disease phenotype is associated with the variant inherited from a specific parent

• ex: UBE3A → angelman

explain angelman syndrome

• UBE3A is methylated (silent) in father, but UBE3A gene is unmethylated (expressed) in mother

• if mom’s is mutated/methylated there is NO UBE3A gene

result = angelman syndrome

what is interesting about prader-willi and angelman

mutation of the same chomosome 15

• pws = mutation from father

• as = mutation from mother

What is Uniparental Disomy (UPD)

• two copies of a chromosome, or part of a chromosome, from one parent

• and no copies from the other parent.

what are epigenetic mutation

disruptions in the regulation of epigenetic silencing

what are secondary epimutations v primary

secondary epimutation caused by real genetic mutation

• DNA mutation

• ex: mutation in a gene that regulates chromatin

primary epimutation that is Not caused by a DNA mutation

• ex: improper methylation of a gene

what are 2 tools and databases for epigenetic

1. epigenomics roadmap project

2. encyclopedia of dna elements

what is functional genomics

study of how those dna seq behave in real biological systems

• methylated dna is transcriptionally inactive/active

• unmethylated dna is transcriptionally inactive/active

• inactive

• active

methods to identify region of methylated v unmethylated dna

bisulfite sequencing

• Unmethylated cytosines → uracils

• Methylated cytosines → stay as cytosines

tell me about bisulfite treatment

detection of methylated or unmethlated dna

1. extract dna

2. bisulfite treatment

3. PCR amplification & sequencing

4. C stays a C, it was methylated. C becomes a T, it was unmethylated

1 pro and con of bisulfite sequencing

• detects methylation at both CpG and non-CpG sites.

• reducing sequence complexity, which can make it difficult to create alignments

what is microarray hybridization and what does it do

• comparing 2 dif samples to detect differences

• 2 dif samples of DNA or RNA are washed onto a hybridized chip. compare how strong the sample binds to the probe via dif colors.

method to detect regions of condensed chromatin v uncondensed chromatin

• atac seq

• DHSS

what is atac seq and explain

• method for detecting open chromatin

• pro: simple and fast sample prep

• fresh tissue isolation

when reading the map of HSS and atac seq, what do the peaks indicate?

open chromatin

what is DHSS and explain

DNase Hypersensitive Site Mapping

• dhs is site where dna is not wrapped around histone tightly

• this means DNase I can access it and cut it

pro: great sensitivity at promoters

con: time consuming

methods to identify dna sequences at which specific proteins are bound

1. chip seq

2. cut and run

3. cut and tag

chip seq

method to find binding sites for specific TF

• crosslink dna

• digest

• add antibody of interest

• capture and wash the complex

• undo cross link and seq the purified DNA

what would a peak from chip seq indicate

there is an occupied binding site

how could you use atac seq, hss, and chip seq together

• atac seq and hss to identify areas of open chromatin

• chip seq to determine sites that a protein binds to

• they should both be the same region

methods for genome wide measurement of gene mrna expression

1. microarray

2. rna seq

what would you use rna seq for?

   Use to compare gene activity

• in different cell types

• in disease vs healthy cells

• under different conditions

cancer is a heterogenous group of disorder. what does this mean

Each cancer type has its own causes, mutations, and behavior (breast cancer ≠ leukemia ≠ melanoma)

yet they share 2 common properties

1. uncontrolled cell proliferation (divide and grow)

2. cell spreading to nearby tissue

hyperplastic v dysplastic growth

hyperplastic: excessive number of cells that appear the same a those in normal tissue

dysplastic: growth that has abnormal cells

benign v malignant tumor

benign: no invasion, self limiting (absense of blood vessels limit size), can be removed surgery

malignant: tumor spread to neighboring cells, grow fast

what does the darwin model of tumor progression say

Tumors are not just masses of cells — they are dynamic, evolving populations of cells that accumulate mutations, compete for survival, and expand when advantageous mutations arise.

2 consequences

1. clonality tumors descend from a single ancestral cell

2. genetic heterogeneity = cells are dif and respond dif to treatment

2 types of cancer genetics

• familial = hereditary = germline mutation

• sporadic = not hereditary = somatic mutation (not germ cells)

characteristics of fam pedigree with sporadic cancer

1. few affected fam members

2. onset late in life

2 types of cancer related genes

proto-onco gene: promote cell proliferation and survival / dominant oncogenes

tumor supressor: prevent inappropriate cell proliferation or survival / recessive oncogenes

gain of function mutation v loss of function

gain: causes a gene product to do more than normal

loss: reduces or eliminates the normal activity

what type of mutation in proto onco genes would promote cancer

gain of function

3 ways that proto onco genes can be activated

1. mutation within the gene

2. multiple copies of the gene

3. gene moved to new dna locus = under new control

promoter fusions

activation of proto-oncogenes

• occur because oncogene is placed next to a strong promoter or enhancer

ORF fusions

activation of proto-oncogenes

• occur because orf of 1 gene is fused to another gene that is already highly expressed

what is interesting about cancer

• recessive at the cellular level, (tested by the heterokaryon test)

• but the inheritance pattern is dominant

2 hit hypothesis

• heterozygous person (but norm func)

• but only needs 1 more mutation event to lose Rb function completely (loss of heterozygosity)

• then the cell gives rise to a tumor

dominant negative mutation

produces a mutant protein that actively interferes with the normal, wild-type protein.

reasons for dominance of oncogenetic loss of function mutations

1. dominant negative effects

2. haploinsuffiency

3. loss of heterozygocity

haploinsuffienciy of some tumor supressor genes

loss of 1 of the 2 functional alleles is not enough to maintain normal cell function.

what are dominant negative alleles

Dominant-negative alleles antagonize the function of the wild type allele

• similar to the dominant negative effect

• 1 rotten apple ruins the bunch

heterokaryon test

test used to determine if a disease is caused by a dom or rec mutation.

fighting cancer the old way and it’s challenges

1 - surgery, radiation, chemo

2 - effectiveness (new cancer from therapy, incomplete removal), side effects (target only cancer)

target cancer drug

act on specific targets - likely proto oncogenes because inhibiting them should slow down growth of cancer

criteria for picking targets (using the targets treatment approach)

1. area with high expression of the protein

2. missense mutation (snp)

3. gene fusion

car t therapy for cancer

• a person T cells are genetically modified to express a CAR

  • car = synthetic receptors that make the t cell recognize specific cancer markers

• CAR T cells seek out and destroy cancer cells

ex: KYMRIAH

risk of car t cell therapy

• neurological toxicity

• cytokin release syndrome

limitation of car t cell therapy

• highly specific antigens are not available for most cancers

• sometimes attacks the healthy cells

  • work around: double gated car t: car t cell only becomes activated if 2 antigens on tumor cell match.