genetics-midterm 1

Blending of traits

The organisms has a blend of values of their parents trait

Partial/incomplete dominance

Heterozygote displays an intermediate phenotype between both of the homozygote parents

HW principle definitions + criteria

allele frequenices remain in equilibrium/constant in a population over generation. If certain conditions are met

• population size is large

• no new mutation

• random mating

• no migration

• no selection

• also no bottlenecks/founder effects

what are the biomedical implications of microevolution

genetic disorders: prevalence of genetic disorders within a population

genetic resistance: genetic variants may confer resistance to specific diseases

drug resistance: development of drug-resistant strains of pathogens

What is the difference between sickle cell disease and traits

disease= homozygous for both

trait= carries allele but don’t manifest symptoms

What questions would you ask to determine is Gabriel has sickle cell disease?

• Blood smear- can view the structure of rbc

• Electrophoresis- can see diff. hemoglobin migration

• Hemoglobin count- for ex: hemoglobin-s

how many cases of sickle cell disease as of 2020?

7.74 million cases

Malaria endemicity

• The prevalence of sickle cell disease coincides with malaria endemicity

• malaria is caused by 1 of 5 parasite (plasmodium falciparum is the most common)

what mutation causes sickle cell

• mutation in the beta-globin gene (HBB^s allele)

what is the pathophysiology of sickle cell disease?

• HbA (normal B-globin gene)

• HbS (mutated B globin gene)

• Normally it has 2 alpha and 2 beta sub-units that are normal

• but in sickle cell there is a mutation in the beta-globin gene of the b-subunit (so it is called hemoglobin S)

  • this mutation causes thymine instead of adenosine (and valine instead of glutamine)

Why does sick cell protect against malaria?

• Malaria normally increases the expression of PfEMP1 that binds to ICAM1, which then allows infected RBCs to stick to the surface and refuse to be removed by the spleen

• Infected individuals have less PfEMP1 expression, which means fewer infected RBCs stick to the blood vessels.

  • increased immune clearance

  • less pathogen maturation

  • reduced endothelial adhesion

• also more free iron and heme so more CO2, which helps tolerance to the pathogen

• interference with actin dynamics on the surfaces of the rbc membrane can cause reduced inflammation

why does HBF help relieve symptoms of sickle cell

• HBF only has alpha and gamma subunits

• This means that there is no beta subunit/beta globin gene, so no HBS polymerization

• The gamma subunit is repressed by BCL11A which then activates the beta globin gene

• symptoms of sickle cell only appear once BCL11A is expressed (usually after 6 months of age)

DNA polymorphism

alternative forms of a genomic locus that differ in sequence or length

SNP

single nucleotide polymorphism

SSR

a short repeating sequence that can repeat a certain # of times

VNTR

variable number of repeats of a long sequence

RFLP

A restriction enzyme that cuts DNA sequences into fragments

• exogeneous

how are DNA polymorphisms used as DNA markers

• dna polymorphisms can be used as DNA markers that narrow down the location of a disease

• genetic linkage means that certain regions on sequences that are close together are inherited together

  • if a dna marker is inherited with a disease it may contain a disease causing region nearby

Haplotype

A series of SNPs that are inherited together due to being close together and from linkage.

what is the unicentric model

• individuals may have the mutation but the surrounding areas may have different haplotypes (same gene but different surrounding regions)

• the HBs mutation happens on the HBB gene, but surrounding region may vary

• uncentric model states that there is one origin and then it extends from there creating new genetic variants

how did sickle cell possible spread

• gene flow and founder effect

  • transatlantic slave trade (people from africa to america)

local ancestry

• looking for one specific region on a genome that can help point to one specific ancestral background

• for example chromosome 11 has dna that can be pointed back to african ancestry, which is where the HBB gene is located

  • more on americans than brazilians

admixture plot

• an indicator of diveristy of DNA

• ancestral portions of a genome

• uses genetic markers

How did HBS allele persist in Jamaica despite a decrease in malaria

• more fitness so survival till reproductive age so women can pass it onto offspring

• more preventative measures —> palpitations for spleen and antibiotics

• fertility rate is decreased in women now but still the same in women with HbS

where is ben are car haplotypes more popular?

car: brazil

ben: america

what was the fertility rate decrease in the jamaica study

• 58% decrease for normal population

• no decrease for SS women

How to find a genotype

• take WBC (contain nuclei and genomic DNA)

• amplify and fragment DNA

• add in probes called oligoprobes that are found on the glass beads/slides of the assay

  • they are complementary to target strands of known dna sequences

• when they bind the light up

• the oligoprobes end one nucleotide short of the sequences: so then you add in DNA poly and nucleotides and it binds to the last spot (the colour let off matches this)

How are DNA markers used to detect disease

• dna markers are sequences of known location such as snps

• they can then be assessed through genetic linkage and sequencing the linked sequences

• a pedigree could also help look at recombination but it must be large enough

what is the formula for phenotypic variance?

Vp= Vg+Ve

what is the formula for genetic variance?

Vg= Va+Vd+Vi

what type of variance does GWAS focus on?

additive: captures additive variance as these genes are normally polygenic

what are complex (quantitative) traits?

• measurable traits controlled by the cumulative effects of many genes (polygenic) at many different loci

what is a gwas?

• A rapid study of many markers across the genome to find genetic variants (such as polymorphisms, SNPs) associated with a trait

  • used in plant/animal breeding

  • ancient phenotype reconstruction (Cheddar Man)

  • forensic DNA phenotype

  • healthcare to detect, treat and prevent disease

• lots of SNPs used: 600- 900

Explain the linear regression line

Y= bX+ intercept

Y= trait

b= effect size

X+ # of alleles

intercept= phenotype when zero alleles are present

done for each variant so there many be millions —> does not ensure significance so p value must be included

manhattan plot

• It converts p-values into -log10 values meaning a really long peak means that it is more significant

• the towers may show clumps due to linkage disequilibrium due to many SNPs being connected to the causal one

• each dot= 1 snp

what is the threshold for the manhattan tower for significance

• must stricter threshold (5 × 10^-8)

• this is =7.3 in terms of -log(10)

QQ plot

• it compares p values to predicted p values

• on the line means consistent with chance

• early life-off is due to issues with bias (false positives, population stratification)

• late life off means consistent with genetic associations/ a true signal

Population stratification

• can show an association when it doesn’t really exist

• systemic differences should be corrected by gwas such as ancestry

• for example bagpipe allele G however, cultural factors were not included such as Scottish use of bagpipes

why is ancestry difficult to account for

• not visible

• admixture

• local structure can vary due to relocation within regions leading to different variations of genes

How to account for ancestry and population stratification

PCA: combines many correlated variables into a few larger continous variation that capture the largest variation

what happens after GWAS

finemapping: figuring out casual SNPs using association strength, CD structure and annotation structure.

snp enrichment: finding the region the casual snp is located such as the cell/tissue —> can help figure out cell type to focus on

colocalization: see if SNP is correlated with gene expression by comparing GWAS data to eqTL (expression data).

fetal hemoglobin connection to GWAS

2007- GWAS helped link fetal hemoglobin to BCL11A

2008- BCL11A was linked to repressing fetal hemoglobin

2023- casgevy approved CRISPR for BCL11A

How was the CRISPR done for BCL11A

collect: Sc collected to created more rbcs

edit: The ethryoid enhancer region was repressed using CRISPR-CAS9

return: chemotherapy to prepare bone marrow and then the sc was returned back

result: repression of BCL11A increased fetal hemoglobin production and eleviated symptoms

PGA

• to determine genetic predesposition

• PGA= effect size x # of allelles present

R² (Coefficient of determination)

measures how much of the variance is captured by the model used/predictor

h² (narrow sense hereditability)

• h²= Va/Vp

• measures how much of the variance is due to genetic variance

what is chromatin

repeating units of nucleosomes

what is the difference between euchromatin and heterchromatin

euchromatin: beads on a string —> 11nm and active

heterochromatin: tighly packed fiber —> 30nm and inactive

6ft condensed due to packaging

histone structure

• histone octomer core: 2 sets of 4 protein (h2a,h2b,h3,h4)

• H1= linker histone that holds the nucleosome bead together

• each histone has disordered flexible N and C termini (histone tails) which can be post-transitionally modified

• the core is much more ordered

• the combination of modification= histone code

what do HATs do

• they are used in acetylation

• the neutralize the charge on lysine which then makes the chromatin more accessible and open to transcription factors

• HDACS reestablish the charge

what do the methylation of cytosines means

• methylation of cytosines are normally adjacent to guanines

  • these found in clusters are called CpG islands

  • the methylation of non-cpg sites can also occur

• methylated cytosines normally mark transcription inactivation

• therefore DNA methylation is almost always a mark of transcriptional silencing

what does DNMTS do for dna modication and Mecp2

DNMT 3 & 4: add methyl groups to cytosine

DNMT 1: maintain methylation patterns

MeCP2: they detect methylation and recruit proteins to promote the formation of heterochromatin

what does chromatin remodelling do?

• increases exposure to promoters which makes the chromatin available to DNA TF and DNase cleavage

What are the enzymatic complexes that help chromatin remodelling

SWI/SWF: they both help slide or eject histones

H3.3 and H2A.Z: they replace histones with ones that promote transcription

what is the ATPase translocase domain

• located on the most modellers that can be interrupted by insertation

• it drives the movement of DNA relative to histones

• important in remodelling

• surrounded by other histone modifiers, ATPase regulators and chromatin regulators

remodellers have 3 main functions

• assembly/organization: ISWI and CHD

• chromatin accessibility: SWI and SWF

• histone editing: H3.3 and H2A.Z

how does histone sliding work?

• the translocase domain is made up of 2 subdomains: the torsion and the tracking

• the torsion binds to dna

• the tracking binds to histones

• the ATP hydrolysis helps the formation of a loop in the DNA gyre and this loop goes around the nucelosome to disrupt the histone/dna interactions

CHIP sequencing

• able to tell what DNA are associated with the proteins of interest

1. crosslinking- freeze the protein in place

2. fragmenting- break the DNA in fragments

3. immunoprecipitation- add antibodies so dna bind and move through genome

4. extract protein and dna

5. dna analysis: then separate the dna to the protein of interest

DNase sequencing

• DNase cuts the sequences and then the cuts are connected back to the genome to identify regions of chromatin accessibility

• the chromatin accessibility regions are called DNase hypersensitive sites

Bisulfite sequencing

• turns all unprotected cytosines into uracils

• then read through PCR

• the PCR reads the U as a T

• compare the U and the T’s

Intergeneration inheritance

• epigenetic inheritance and the mark is passed on to offspring of the individual directly exposed to the stressor/environment

transgenerational inheritance

• the epigenetic inheritance is when the mark is maintained even when the offspring is not exposed to the stressor/environment

• for transgenerational it depends on the gender

  • women pass on trauma to all of her eggs so we look beyond F2 generation

  • for males you look beyond F1 generation

  • if mark is there past these then it is transgenerational

Methylation maintenance

• DNMT1 sees the markers on the hemi-methylated DNA and uses this as a template to add appropriate methyl group to cytosine

• there are also histone modifications: these are histones that are from broken down nucleosomes and are evenly distributed to daughter strands

• can also be maintained during gametogenesis and fertilization and then carried to zygote

C value paradox

disconnect between complexity of an organism and its genome size

• human have 3.2 million bp and then amoeba are more complex but 600 billion bp

G value paradox

disconnect between complexity and protein coding genes

• humans and C.elegans have the same number of protein coding genes —> despite differing in complexity

what can contribute to the G & C value?

• alternative splicing and gene expression regulation

what are the two types of non-coding RNAs

housekeeping and regulatory

explain the housekeeping ncRNA

trna: transfer animo acids

rrna: help ribosome function

snrna: helps cut out introns in mrna

snorna: helps modify and process rna

explain the regulatory ncRNA

lncdna: gene expression and chromatin structure

mirna: helps decrease protein function through target mRNA

sirna: silence genes through target complementary DNA

pirna: helps remove transposable items such as in germ cells

what define long and short ncdna

long: greater than 200 nt

short: less than 200 nt

biogenesis of lncRNA

• transcribed by RNA poly II

• has poly-a-tail and methyl cap

• has few exons compared to mRNA

• does not translate into proteins

• it can bend, loop, dent etc. which controls what proteins the lncdna can interact with

colocalization

• stays in the nucleus or cytoplasm

nucleus:

• due to chromatin degradation, chromatin retention, weak splicing, and binding to nuclear proteins

cytoplasm:

• lncRNA can also stay in the cytoplasm

• finds ribosomes, locates mitochondria or associates with exosomes or other organelles

Xist- an example of gene regulation

• xist explains how x-inactivation occurs

• it shows how there are 4 repeats in the xist gene (A,B,C,E)

• A: it controls gene expression by using PCR 2 to add on H3K27ME3

• B: it also controls gene expression but by using hnRNP-K and PCR1 that adds on H2A117UB

How are small non-rna produced (SiRNA and MiRNA)

SiRNA:

• made from dsRNA (a sense and antisense strand)

• dicer —> risc/ago complex—> mRNA

• dsrna is made from cisrna (overlapping regions of two strands)

• it is exogenous, 23-27nt long, made from rna poly III and precursor is dsRNA

MiRNA:

• made from single stranded rna that loops around

• drosha to cut ends —> dicer —> risc/ago —> mrna

• endogenous, 17-23nt, made from rna poly III and precursor is the hairpin loop

QPCR

• measures RNA abundance using RNA turned into CDNA

Reporter assay

• uses a signalling molecule: lucifrase

• it then added in the 3’ UTR sequence which has binding site

• if it binds then less light produced by lucifrase

pm2.5

• particulate matter that is 3.5 uM or smaller

• reaches alveoli and bloodstream

• causes respiratory, cardiovascular and neurological diseases