Discovering Mutation Rates

• Distinguish between the meaning of deletions, insertions, frameshift, missense, nonsense, and
synonymous mutations.
o Synonymous mutation: Residue is substituted with a similar one. Usually doesn’t result in a
significant change in protein function.
o Frameshift: Addition/excision of a single bp alters the entire reading frame of a gene, often
resulting in early stop codons.
o Indels: Addition or removal of bps from the DNA. Often frameshift but can also eliminate or
add entire residues on a protein.
• Does clonal evolution in cancer happen with somatic mutations, epigenetic changes, or both?
o Evolution in both occur in cancer. This contributes to the overall diversity of cell lines.
o Most undergo bottleneck effects over the course of treatment, selecting for those which
can survive.
o Methylations can shift in cancers. Common in gliomas, leuks, colon cancer. Epialleles can
also be selected for, just like specific mutations.
• Are most cells in the same cancer identical?
o No, most cancer cell lines contain a unique set of mutations that differ notably from
progenitor populations. More lineages arrive as the disease progresses, enabling
• What are non-coding genes?
o Genes encoding a transcript that is not translated into a protein. Rather, the RNA itself has a
function, eg. rRNA, Xist, Tsix.

Genetic Discrimination, Molecular Privacy, and Designer Genomes

• What are the protections offered by GINA legislation in the US? What does it NOT cover?
o Protections:
§ Health insurance issuers from discriminating against individuals in eligibility,
coverage, underwriting, premium-settings
§ Employers can’t hire, fire, promote, pay or assign jobs based on genetics
§ Employers and related entities like unions can’t request genetic information
o Not covered:
§ Companies w <15 ppl
§ Military
§ Long-term care, life, and disability insurance
• What are the three requirements for a patent to be valid in the US?
o 1. New – “some new entity that never existed before,” like a cDNA, not your genome
o 2. Useful – has to be relevant toward something.
o 3. Non-obvious – some vague idea relating to the gene rather than the actual gene itself.

Cancer Genomics***
• Cancer = genetic, rarely hereditary, old-age monoclonal disease. Telomeres, angiogenesis, multi-
step process
• Distinguish between proto-oncogenes and tumor suppressors. Which are usually mutated in
hereditary cancers?
o Proto-oncogenes are genes that, after a GOF mutation, go on to cause cancer
o Tumor suppressor genes protect us from cancer by inhibiting uncontrolled cell growth. They
can 1) directly guard, 2) be caretakers (eg, p53), or 3) be related toward the cell cycle (eg.
Telomerase).
o Tumor-suppressor genes are usually hereditary, because predisposition to a cancer can
follow an autosomal dominant fashion. Oncogenes, in comparison, are often generated
from single somatic mutations.
• What are three ways that a proto-oncogene can become oncogenic?
o Point mutation, oncogene amplification, chromosome translocation. They all usually act
dominant.
• What is the Knudson two-hit hypothesis, and how does it apply to retinoblastoma?
o Hypothesis that tumor suppressor genes require mutations within both alleles in order to
enable cancer.
o Explains why predisposition to somatic cancers is autosomal dominant, yet recessive at the
cellular level as the first hit is merely hereditary from parent to child.
o First hit is usually a point mutation, second is point or larger.
• For each gene below, describe 1) the ways each of these genes can contribute to or prevent cancer,
2) the particular cancers they are associated with, if known; and 3) other names for structures or
mutated proteins associated with this gene, if any:
o Ras
§ 1) Ras signaling is involved in promoting cell division. Mutation (cd12 Gly-Val) blocks
GTPase activity, keeping it stuck in GTP-bound active form.
§ 2) Human bladder carcinoma.
o Myc
§ 1) Myc proteins induce factors in cell cycle, protein synthesis, and a lot of other
things necessary for division. Uses oncogene amplification by doing duplicating a lot
or (creating homologous staining regions) or generating double minutes.
§ 2) Varies based on the myc mutated. Eg. N-myc causes neuroglioblastoma.
§ 3) Double minutes are extrachromosomal structures which contain the myc gene,
used to increase overall gene expression at the transcriptional level. Homologous
staining regions are chromosomes whom, when stained with Giemsa/others, appear
larger than their homologous counterpart due to duplications. Both can be observed
by FISH.
o Bcr-Abl
§ 1) t(9,22)(q34,q11) generates recombinant bcr-abl gene, a Tyr/Ser/Thr kinase which
causes abnormal adhesion, proliferation, and differentiation.
§ 2) Chronic myeloid leukemia
§ 3) Philadelphia chromosome = chr 22 with bcr-abl. Bcr is normally on chromosome
22, while abl is on 9. Can be observed by FISH.
o Retinoblastoma – Gatekeeper TS at 13q14
§ 1) Responsible for directly repressing G1-S transition. LOF = promotes unregulated
proliferation = cancer
§ 2) Retinoblastoma cancer. It’s hereditary (40% cases) or sporadic (60% cases).
Autosomal dominant in predisposing you to cancer, but recessive at the cellular level
because of Knudson’s 2-hit hypothesis.
§ 3) All bilateral eye loss = hereditary, 15% sporadic too. Bilaterals are 30% of cases
overall. 85% of sporadic cases are unilateral (70% of cases overall).
o BRCA1 / BRCA2 – Caretaker TS’ at q17 and q13 respectively
§ 1) At DNA damage in G1/S phase, they help repair dsDNA breaks.
§ 2) Breast cancer – 5-10% of total cases
o P53 – Caretaker at 17p arm
§ 1) TF regulating cell cycle progression + mitosis entry
§ 2) Li Fraumeni syndrome – premenopausal breast cancer, sarcoma, brain tumors,
leukemia/lymphomas, adrenocortical carcinoma. Lost in 50% of cancers
o ATM – recessive gene predisposing to cancer
§ 1) ATM kinase phosphorylates p53 in response to damage, being responsible for cell
cycle stopping. LOF prevents this happening, allows more mutations to accumulate
over time.
§ 2) Breast cancer in carriers, Ataxia Talangiectasia
 o Telomerase
§ 1) Adds/maintains TTAGGG of 15kb to ends of DNA to avoid losing 35bp DNA in each
replication. Cells stop dividing w broken telomeres, so cancers need this gene.

Clinical Sequencing
• Why does next-gen sequencing usually have to cover the same nucleotide multiple times (i.e., why
is high coverage important)?
o High coverage is important bc not all bp’s in DNA are equally accessible by DNA polymerase
during sequence (eg. Due to secondary structures or some fragments randomly being more
common than others). A high coverage ensures DNA pol doesn’t miss a specific sequence.
• How can next-gen sequencing be used to determine whether a patient is homozygous or
heterozygous for a given SNP?
o This is a process called genotyping. All of patient’s contigs containing a specific region/gene
of interest are aligned and compared to one-another. If both maternal and paternal alleles
have a specific SNP compared to a reference genome, the individual is homozygous for an
abnormal SNP. If only 1 chromosome has it, they are heterozygote.
• How does exome sequencing differ from whole-genome sequencing? When is it preferred? When is
whole-genome sequencing preferred? Under what circumstances is array genotyping preferred?
o Exome sequencing observes the transcriptome – it sequences all available RNA’s within a
given cell, rather than its genome. A cheap process best used for screening Mendelian
disease because those are related to one gene we can look for.
 o Whole-genome sequencing monitors everything, which is good when you really need a
reliable sequence or require all regulatory elements within a gene (since it also takes non-
coding sequences like enhancers)
o Microarrays are the cheapest option available, using DNA hybridization to identify genotype
in a large sample size of individuals. Best in ancestry/population studies.
• How can next-gen sequencing be used to detect cancer? What is personalized medicine, and how
does it relate?
o Some tumor cells are constantly circulating within the blood. Taking a sample, doing whole
genome sequencing, and comparing to a reference genome can enable you to find a
proportion of contigs that corresponds to an activated oncogene/LOF TS. If this population
of cells is increasing over time, then it indicates cancer.
o Personalized medicine is the process of tailoring specific treatments toward patient
genotypes. Sequencing can identify the observed mutations within a cancer cell population
– genotyping it- and we can provide appropriate drugs for those defects.

Personal Genome Interpretation

• How might ancestry and consanguinity information be clinically useful to a patient?
o Various populations have different allele frequencies for various diseases, so ancestry can
put forth specific disease the patient might want to get screened for. Eg. Ashkenazi Jewish
ancestry would indicate Tay-sachs, Canavan, etc.
o Consanguinity results in increased homozygosity within the patient, which heightens the
chance of autosomal recessive Mendelian disorders. Can be measured through long runs of
homozygosity.
• How can studying protective and deleterious mutations aid in understanding a disease?
o Observations of these mutations can identify targets for therapy.
o Extreme sequencing – doing it on individuals with the strongest forms of specific traits – is
used to identify protective and deleterious mutations.
• What are human knockouts? How are these individuals identified?
o Human knockouts are individuals who have no functional copies of a specific gene at EVERY
CELL, unlike in cancers (somatic mosaicism). From double LoF mutations. Some are not
possible bc gene is essential
o They are found by reverse phenotyping some biobanks, a process termed biobank mining.
One individual containing the LoF mutation is first identified through the phenotype, after
which their lineage and ancestry is observed to see if anyone related is affected. If they are,
you can observe for the gene in the rest of the human population to observe its prevalence
o This process can identify some new variants, as now you can observe for its effects on the
body
• Which are the most important kinds of variants to look at first?
o Usually prioritize variants within the exome because they are most associated with
phenotype through the proteins they encode. Within those are 3 categories:
o Variants with known links to phenotype are best studied and associated with various patient
outcomes. Because they are most likely to impact the patient’s life, these should be
prioritized.
o Next comes variants of genes with known links to phenotype. These aren’t as important
because while our gene is relevant, it is uncertain whether or not the specific variant causes
a difference in phenotype.
o Least important are variants computationally linked with a phenotype. These may have
come from sharing haplotypes with other genes and could be coincidental. They have no
characterized mechanism of action, and so are very unlikely to cause anything in a way we
can understand.
• What are some tools you might employ to study the relevance of a variant to a given disease?
 o We identify relevant variants through a combined computational/manual approach
o Can do variant function predictions:
§ 1. categorize based on known literature
§ 2. conduct location annotation (see if it’s in the coding or splice sites which often
cause changes rather than just intergenic spaces). 95% are usually non-coding.
§ 3. screen for evolutionary disruption to see if the coding variant is disruptive – which
is the case 22% of the time. Afterwards do the next steps below.
• Polyphen can help here
o Observe variant frequencies – common diseases have common variants, opposite for rare
diseases. The variant’s frequency should match the phenotype at this respect
§ ENSEMBL can show the variant’s freq
§ They need to follow Hardy-Weinburg expectations
o After that, you can go to variant databases like Clinvar, OMIM, etc to see if the variant is
known linked with a specific phenotype.
§ OMIM

Treatment of Genetic Disease***

• What are some genetic diseases that can be managed with lifestyle modifications?
o Hypertrophic cardiomyopathy – Avoid strenuous physical activity. Left ventricle outflow
obstruction.
§ 1/500 carriers. 27 genes involved
o PKU – Restrict Phe in diet
o Maple syrup urine disease – a-kg DH deficiency, avoid Val, Ile, Leu
o Ornithine trancarbamylase deficiency – Limit NH3 production (eg. Less meat)
• Describing CF + Sickle cell anemia:
o Cystic Fibrosis:
§ Usually causes chronic bronchitis + pancreatic insufficiency (latter is saved by
enzymes).
§ Neutrophils come in, explode. Makes bronchioles thicken too much and destroy
alveolar parenchyma.
§ CFTR – cAMP channel. On apical membranes of exocrine epithelia + airways.
Dysfunction = limits Cl- movement.
§ 1000s of mutations
o Sickle Cell: beta-chain abnormality.
§ Commonly 6Val->Glu.
§ W low O tension, cells change shape. Disrupt capillaries, and RBCs have too low half-
life.
§ Infarction of bone, spleen, etc. Latter gets destroyed a lot and you get susceptible to
infection
• How can Cystic Fibrosis and Sickle Cell Anemia be treated with small molecules?
o CF: Can treat G551D variant with Ivacaftor, which increases Cl- movement
o Sickle cell: Hydroxyurea activates fetal gamma hg to substitute for b-chain.
• What is augmentation therapy? Give examples of two genetic diseases that can be treated this way.
o Process where we administer a missing/weakly expressed protein to a patient to alleviate
disease symptoms.
o Mostly comes from pooled plasma or recombinant proteins
o Hard to get proteins into the cell though – eg. Brain/eye barriers
o Eg. 1 - Main example is a1-antitrypsin deficiency.
§ Second most common blood protein, associated with emphysema/asthma/liver
disease.
§ 1 mutation – Neutrophil elastase Z mutation – Glu -> Lys
 § Destroys parenchyma of lung
§ <11uM = threshold for this
o Eg. 2-4: Other protein-related ones:
§ Hemophilia A/B (factor8/9)
§ Hereditary angiogema – C1 esterase
§ Hereditary immunodeficiencies.
• How do proteins to treat lysosomal storage diseases get into the cell compartment where they can
be effective?
o Lysosomal diseases are from accumulation of waste products, often autosomal recessive.
50% CNS, mostly fatal, heart skin kidney and joints.
o Enzyme augmentation therapy used to supplement weakened enzyme.
o These use mannose receptors to get into cell – add mannose receptors, causes receptor
mediated endocytosis.
o Might worry about immunity against protein or compartment-specific disorders (eg. Liver-
specific, you don’t want it to go elsewhere).
• Which organisms are used in delivering gene therapy? Which classes of this organism are used?
o Viruses - Adenoviruses adeno-associated viruses, retroviruses, lentiviruses, herpesviruses
o Some are retro, some are dsDNA
• What are the risks of doing gene therapy?
o You can build up immunity which can result in aversive reactions or weakening overall
treatment, leading to death (eg. Gelsinger case).
o Retroviruses can insert into genome – if they turn on an oncogene they will cause cancer.
• What are the advantages of doing in vivo instead of ex vivo genome editing?
o In vivo: They are needed in proliferating targets bc they integrate into the genome and
therefore get placed into all subsequent cells.
§ Mostly uses lenti (best one) and retroviruses.
o Ex vivo: Non-proliferating cells. Eg. Heart, muscle.
§ Adeno-associated viruses.
• What are the major risks of using retroviruses and lentiviruses for gene therapy? What are their
advantages over adenoviruses?
o Retro/lentiviruses use reverse transcriptase which can cause insertional mutagenesis into
promoters and disrupt transcription. Very common in gamma retroviruses.
§ You need to also make sure they don’t replicate which makes them pathogenic
o Sometimes we deplete the bone marrow, modify it ex vivo w this and re-establish immune
system – can cause infection.
• Why are adenovirus vectors considered more safe? What other advantages/disadvantages are
there in using AAV instead of retro/lenti-viruses?
o Don’t modify nuclear DNA so no insertional mutagenesis
o Eg. LINCL.
o +s:
§ Can modify cell tropism with capsid
§ No mutations
o -s:
§ Only 1.5kb capacity – really low. Bad for large genes
§ Immunity can rise against capsid
• General issues w viruses:
o Manufacturing the protein
o Delivery – how do you get enough?
o Specificity, off-target effects
• Gene therapy through these viruses can repair any gene-mediated disorder. Your choice of virus
depends on the system you need to fix – replicative = retro, non-replicative = adenovirus