Population

Genetics
• In a population with 60% AA genotype, 30% Aa genotype, and 10% aa genotype, what is the
frequency of each allele (A and a) in the population? Is it in Hardy-Weinburg equilibrium?
• 60 + 30 + 10 = 100
• F(a) = ½*0.3 + 0.1 = 0.25
• F(A) = 0.6 + ½*0.15 = 0.75
• Not in equilibrium bc f(a)*f(a) doesn’t equal f(aa).
• What assumptions must be satisfied for Hardy-Weinburg equilibrium to hold?
• 1. No Mutations
• 2. No migration
• 3. Random mating
• 4. Very large population
• 5. No selection pressures
• If I know that a disease exists at a frequency of 1 in 5000 persons, what is the percentage of carriers
of the disease in the population? What if the disease occurs for 1 in 10 people?
• 1/5000: p is about 1, so 2pq = 2sqrt(1/5000) = 0.028
• 1/10: q^2 = 1/10 = 0.1 = 10% -> q = 0.01, p = 0.99 -> 2pq = 0.188
• How do consanginous marriages & assortative mating affect Hardy-Weinburg equilibrium?
• Consanguinity and assortative mating favouring similar individuals will increase the rate of
homozygosity. If assortative mating favours opposite pairings, it will increase heterozygote
frequency.
• What are the bottleneck and founder effects?
• Founder: When non-random (eg. Non-representative) sample of a population establishes
a new population. The new one will have different allelic frequencies from the old
• Bottleneck: When a selection pressure alters allelic frequency by killing off those of a
specific phenotype, allelic frequencies among the remaining population is different from
the original.
• Both are examples of genetic drift, when non-random event causes a change in allelic
frequencies over time.
• Why do dominant diseases often not have Hardy-Weinburg equilibrium?
• Individuals homozygous in a deleterious dominant allele are often selected for, meaning
they do not exist. Because q^2 = 0, while q > 1 (as heterozygotes exist), there is no
possibility to reach a Hardy-Weinburg state.

Polymicrogyria = abnormal layering of cerebral cortex

Epigenetics
• What is epigenetics?
• Study of inheritable features not encoded by DNA bases. This can include DNA
methylation or nucleosome tail alteration.
• What is a nucleosome? How is it connected to gene expression?
• Nucleosome is an octamer made of 2 each of H2A, H2B, H3, H4. H1 is a connecting
protein. Alterations of histone tails can alter gene expression by changing its overall
charge, which would increase/decrease its affinity to DNA.
• How do histone acetylation changes lead to changes in gene expression? Do they usually increase
or decrease gene expression?
• Acetylation generally decreases + charge, which would make it wrap less tightly ->
increase expression. Methylation generally decreases expression.
• In what environment do we usually find methylated cytosines? Does this generally lead to
increased or decreased gene expression?
• CpG islands are sites of CG repeats on a chromosome. Methylation of cytosines
by DNMT will decrease gene expression.
 • How is methylation status maintained when the DNA is replicated?
• DNA is hemi-methylated post-translation. Maintenance methylase theory hypothesizes
the enzyme DNMT uses hemimethylated sites to know where to methylate CPG, though
this is still unconfirmed.
• How does DNA methylation lead to Barr bodies, mosaicism, and calico coloring in cats? Describe
the interactions between Tsix and Xist.
• Barr bodies = inactive X chromosomes, usually bc only 1 is enough for a cell to do its
functions
• X chromosome methylation in a cell is governed by dichotomy of Xist and Tsix: Xist is
transcribed in cis, generally recruits DNMTs to methylate DNA, and deacetylates
histones. Tsix is an antisense RNA that counteracts Xist, which allows Xa to stay active
• The X chromosome which dominates in a cell line (eg. Paternal or maternal) is
decided very early on in a calico cat’s development. All progeny in each subset will
subsequently express the same Xa as their ancestor. Following this, three regions of
the calico cat are made: 1) place where they’re supposed to be no colour; 2) regions
where maternal X was activated; 3) regions where paternal X was activated.
• What does it mean to be paternally imprinted for a given gene? What epigenetic mechanism is
used to do imprinting?
• Paternally imprinted means the paternal gene receives a specific epigenetic
modification while it is in the sperm – usually represses transcription. This occurs
before fertilization.
• Imprinting is controlled by ICE/IC/ICR regions (imprint control elements/centers),
which contain 3-11 imprinted genes. These contain CpG islands which can be
methylated to repress downstream genes.
• Distinguish between the molecular mechanisms of Prader-Willi Syndrome and Angleman
Syndrome in the case where both are caused by deletion of the same region of a chromosome.
What other molecular mechanisms can lead to each syndrome?
• Praeder-Willi can be caused by maternal uniparental disomy or defective paternal
alleles within chromosome 15 q11-q13. There are a set of maternally imprinted genes
upstream of the ICR, who are paternally expressed.
• Angelmans uses the paternally imprinted ubiquitin ligase gene, which is maternally
expressed. Needs uniparental disomy from father or defective maternal allele for
disease.
• How can imprinting (or loss of imprinting) be used by cancer for overexpression?
• UPD/mutation for an imprinted tumor suppressor gene will increase rate of cancer as
inhibition is reduced. UPD of a gene from the non-imprinted parent can trigger proto-
oncogenes to full oncogenes.

Genome Editing
• What is the original CRISPR/cas9 purpose in bacteria?
• Adaptive immunity against virulent (eg. Viral) DNA. Problematic genomes would be
integrated within spacer regions, which would be transcribed by the cell and processed
into guideRNAs. With a tracrRNA + guideRNAs, Cas9 would be able to bind anything
corresponding to guide RNAs (from spacer regions) and create a ds break.
• What is the role of the guide RNA in the CRISPER/cas9 system of genome editing
• Guide RNA brings cas9 to complementary DNA, which localizes the nuclease domain so it
can make a ds break. Usually needs a tracrRNA to stabilize non-binding regions, which we
just combine in lab.
• When do we want the cuts made by the Cas9 protein to be repaired by NHEJ, and when do we
want it to be repaired by HR? How do we control with process is used?
• NHEJ: when we want to knockout a gene. Cas9 would cause a ds break, and NHEJ
intrinsically has a high mutation rate because of exonuclease degradation. Repeated
 cycles of this would eventually cause indels within the gene, disrupting its
transcription level or protein function. We can control this by growth arrest (no HR)
+ adding no homologous DNA.
• HR: Same ds break as cas9m, except we can also incorporate a homologous DNA
into the cell. This DNA would be inserted by HR machinery.
• What are the techniques that can reduce the chance of off-target mutations being caused by the
system?
• 1. Introduce RNA to the cell rather than DNA.
• 2. Use nuclease dead Cas9 fused to other proteins like transcription repressors to alter
gene expression, rather than excising DNA itself
• 3. Cas9 nickase + base editing enzyme can modify a bp
• 4. Target RNA rather than DNA using Cas13
• How can this system be used to affect epigenetic modifications?
• Dead Cas9 bound to HDAC/HAT/Tf. Eg.p300 = HAT, LSD1
• Can also add DNMT or DNA methylases too
• How does the Cas13 differ from the Cas9 system?
• Cas13 is an RNAse, which doesn’t make a ds break. Just degrades an RNA.
• Nuclease dead Cas13 fused to base editor can make fixes to abnormal mRNA, fixing its
coding sequences. This lets us fix the cds without altering DNA.
• Distinguish between in vivo and ex vivo editing methods, and the advantages of each.
• These are delivery strategies of therapeutic CRISPR DNA
• Ex vivo editing simply delivers CRISPR DNA/RNA in a lab. They isolate some defective cells
from host, edit them, and graft em back.
• +s: Control over doses of molecules, many delivery platforms to add DNA, high
editing rate bc we choose efficient delivery methods
• -s: Cells need to be able to survive manipulation outside the body – they NEED
stem cells/cancer in their population, grafting can go poorly
• Eg. PD1-L shutdown in tumor cells through CRISPR, or make chimeric T cells
• In vivo methods introduce the DNA in situ. Eg. Viral vectors with specific tropisms, or
delivery agents that target specific cell types.
• +s: Useful for pops that can’t sustain themselves (eg. Retina), can target
multiple organ systems at once
• -s: Largely virus -> can only go to infectable organs like eye or liver, Immune
response (Can avoid with RNA CRISPR or nanoparticles), dosing control difficulty
-> can make off-target mutations.

Complex Disease
• How do we distinguish between simple and complex genetic diseases?
• Simple closely relates genotype to phenotype, complex has poor association
• Complex’s genotype can protect/predispose ppl to disease. They’re multifactorial and
polygenic, mostly prevalent.
• Gene-gene, gene-environmental interactions control it.
• What is the difference between continuous and discontinuous traits?
• Continuous = quantitative, eg. IQ or blood cholesterol, blood pressure
• Discontinuous = categories. Eg. obesity, hypertension, diabetes.
• Why are family pedigrees not usually useful when considering complex diseases?
• They don’t follow any classical Mendelian inheritance. Pedigrees can instead be
misleading and confuse us.
• Explain why polygenic diseases tend to have continuous traits.
• When a trait is polygenic, it tends to have a lot of variation in values between people, as
they all have different alleles that contribute differently to the phenotype – locus
heterogeneity.
• Continuous traits exhibit the most variation, with infinite possibilities between each whole
number. To match the variation from polygenic traits, most of the traits are continuous.
• Eg. The more genes you have, the more possible outcomes. With a lot of genes + alleles,
the outcomes eventually follow a Gaussian distribution.
• How can mono- / di-zygotic twins studies be used to estimate environmental vs genetic
contributions to traits?
• You can compare outcomes in mono and di-zygotic twins to understand
contribution of environment.
• Monozygotic are gnomically identical (except mito) – share 100% alleles
• Di-zygotics are like siblings – 50% identical (R = ½), and also controlled for age
+ environment.
• Higher concordance rates (rate of shared frequencies) in monozygotes vs Di =
genetic influence.
• If MZ concordance < 100% shows environmental influence
• Eg. Depression, reading disability, Alzheimer. Height is 95% in MZ twins, IQ
90%, Diastole BP 50%.
 • Family aggregation + Risk ratio:
• When f(disease) in family is greater than that of general population.
• lr = risk ratio. If 1, no additional risk for disease. Higher = more risk. Replace r depending
on relative
• How do case/control studies differentiate between genetic and environmental factors?
• They match age, sex, environment, and do unrelated cases + controls
• They select specific genes and then observe for odds ratio bw the population
• Odds ratio = expected outcomes according to relationship / not expected
• Eg. Ad/bc

Reproductive Genetics
• Why are older women more at risk of aneuploidy?
o Ovaries select most fit eggs for growth during every period – the ratio of proper eggs gets
lower as you get closer to menopause. Also the rate of non-disjunction in meiosis happens
more often – meiotic spindle becomes more error-prone.
o Birth = 1 million, puberty = 250,000, 38 = 25K, menopause = 1K
• What is the difference between screening and diagnostic tests?
• Screening tests observe for the risk of a disease, diagnostic is used to confirm disease.
• Eg. 9-13 weeks. First trimester combined test – ultrasound + serum tests. bHCG,
PAP-A, and nuchal fold.
• Eg2. 15-18 weeks. Quad screening test – AFP, estriol, inhibin A.
• These can test for Down syndrome + lower the expected odds of it.
• Can combine screening results with other identifying factors like age to confirm risk of
disease – so patient can then see if they want CVS/amniocentesis
• How does cell-free DNA testing of fetal genetics work?
• 9-weeks in, some fetal DNA gets sent into maternal blood from the placenta/chorion. We
can extract this and do in vitro studies.
• Application #1: Epigenetic screening. If fetal DNA is different in methylation from mother,
can isolate it and look for variations.
• Application #2: Satellite marker sequencing to find trisomies. Find paternal and maternal
satellite sequences, and couple to mass spectrometry to learn which one is in excess.
• What are the limitations of cell-free DNA testing?
• Time-limitations – can’t do it early
• High BMI = low fetal fraction of DNA
• Can’t do twins or vanishing twins bc can’t distinguish
• Can confuse mother’s with fetal DNA in donor pregnancies.
• What are the diagnostic tests needed to confirm results from cell-free DNA testing?
• 1) Transverse chorionic villi sampling (10-14 weeks)
• 2) Amniocentesis (13-17 weeks) – takes like 4-6 weeks to get results, so disadvantageous
in terms of time
• 1/500-1/1000 fetal loss
• Both use the ultrasound to guide it.
• CVS can be done earlier, but can’t accommodate placental mosaicism.
• These things can test for single gene disorders or aneuploidies.
• List the ethnicities that are especially associated with Tay-Sachs disease, bloom syndrome, sickle
cell disease, Canavan disease, beta thalassemia, cystic fibrosis, and alpha-thalassemia.
• Ashkenazi Jews: Canavan, Bloom
• French Canadian/Cajun: Tay Sachs
• Caucasian: CF
• Mediterranean: beta Thalassemia
• Southeast Asian: Alpha thalassemia
• African-American: Sickle cell
• What test is most appropriate to use in the case of single-gene disorders?
• CVS and amniocentesis can give enough DNA to screen for lots of disorders like
Huntington or CF. It can also be done through cffDNA testing, but that’s just starting rn so
it’s not the most appropriate.
• What is preimplantation genetic diagnosis?
• One of the recent advances of preventing genetic diseases, by having us select which ones
will be implanted during IVF.
• During IVF, we can look at zygotes pre-implantation. Can do day 3-5 biopsy to do genetic
sequencing and observe for aneuploidy/mutation. Embryo morphology is also a good
indicator of aneuploidy.
Newborn Screening
• Distinguish between true positive, true negative, false positives, and false negatives.
• True positive = affected individual had + result
• True negative = unaffected individual had – result
• False positive = unaffected individual had + result
• False negative = affected individual had – result.
• How is sensitivity calculated?
• Positive-affected / total affected
• How is specificity calculated?
• Negative-unaffected / total unaffected
• How are positive and negative predictive values calculated?
• PPV = Positive-affected / total positive
• NPV = Negative-unaffected / total negative
• Should a screening test prioritize sensitivity or specificity
• Sensitivity bc it allows us to find the ppl who need diagnostic testing

Genome-wide Genetics
• Do GWAS studies typically study monogenic or multifactorial disorders?
• Multifactorial. Can do both though.
• How is the odds ratio defined?
• Odds of individual w risk allele developing a disease compared to those without
• Eg. OR = 2 means twice the risk if you have the allele
• If using linkage analysis, why is it important to study multiple generations?
• It narrows down the regions of pathogenicity, as surrounding loci get changed bw
generations through cross-overs or mutations.
• Do GWAS studies identify pathogenic alleles, unlike linkage studies?
• No. They only find protective/risky haplotypes. Need evolutionary studies to establish
pathogenic.