Wk 6 DNA-Based Genetic Testing, Cancer Gene ID TBL 11 Flashcards
Definitition of Heterozygosity
4 possible genotypes at a single locus:
G:C
A:T
C:G
T:A
2 chromosomes in a cell, one from mother, one from father
-same info = homozygous
-different info = heterozygous
Allele
Labeled A or B (before base pairs were known)
heterozygous = A, B
What happens to DNA breaks that can cause loss of heterozygosity?
double strand breaks -> either become
1. homozygous
2. hemizygous (lose a copy of information)
Explain how DNA replication is supposed to go
- diploid cell w/ chromosome copy from mother and another from father (blue and green) (23 pairs, ex 1)
- S-phase replication of both chromosomes -> 4 copies of chromosome (2 mother, 2 father)
- mitosis: 1 copy of mother and 1 copy of father to each daughter cell (2 daughter cells total that are diploid)
What happens if there is a double stranded break in one copy of a chromosome (ex green)?
- repair can be through homologous repair
- two ends of the break invade the homolog
- can resolve w/ crossing over w/ maternal and paternal arms exchanged OR patch repair
- w/ crossing over (homologous repair)
- mitosis occurs and the distal arm of the chromosome where the exchange occurred is now homozygous for one parents DNA = los of heterozygosity (either all blue or all green at the distal end for all of the loci)
What happens if homologous repair does not occur w/ DS break?
=a checkpoint failure
1. during mitosis, centromeres still do their job
2. the unattached fragment that wasn’t joined to a centromere stays behind at mitotic plate, gets lost and stays outside mitotic/daughter cell
3. One daughter cell gets a normal genome, second daughter cell ends up without any of the info on the lost chromosome fragment, only the one copy for the intact chromosome (in blue)
= loss of heterozygosity and is hemizygous w/ just one copy
Remember
How are tumor suppressors recessive at the cellular level?
Both copies of a specific tumor suppressor gene pair need to be mutated to cause a change in cell growth and tumor formation to happen. For this reason, tumor suppressor genes are said to be recessive at the cellular level. Mutations in tumor suppressor genes are often acquired.
What is a microsatellite?
Small pieces of DNA that repeat
-can be unique to individuals, like fingerprints
What is microsatellite instability?
Microsatellites have many repeats of the same sequence, all next to each other
If the 2 DNA strands are taken apart, it’s very difficult to get them back into alignment in phase
likely to miss out of phase sections b/c base pairs can still align but some bases aren’t paired at all
-when DNA replication fork comes along, DNA polymerase doesn’t notice the missing bases with fewer microsatellites. Mismatch repair machinery isn’t alerted, so end up with 2 strands w/ different numbers of the copy repeats
Differing strand repeats won’t be noticed in later DNA replication
What can diagnose microsatellite instability?
PCR
-can look a regions and find different copy numbers, even within tissues from same person
What defect is microsatellite instability associated with?
Mismatch repair defects
What is a neoplasm?
mass of cells w/ uncontrolled growth
AKA tumor
3 Requirements for cancer growth
- disable apoptosis
- angiogenesis
- overcome inhibitory signals to achieve malignant state
Origin of carcinomas
epithelial tissue (most common tumors)
Origin of sarcomas
connective tissue
origin of gliomas
glial cells of CNS
origin of lymphomas
lymphatic tissue
origin of leukemias
hematopoietic organs: bone marrow, spleen, tonsils, and lymph nodes
What are monoclonal tumors?
tumors are usually derived from a single ancestral cell, making them a single clone
Carcinogenesis
= cancer development
In what type of cells do cancerous events most often occur?
Somatic cells
-frequency of events can be altered by exposure to mutagens
-b/c in somatic cells, are not inherited
In what type of cells do cancerous genetic events occur?
Somatic cells
-frequency of events can be altered by exposure to mutagens
-b/c in somatic cells, are not inherited
When are cancers inherited?
When mutations occur in germline cells
Why are people w/ a germline mutation highly likely to get cancer?
Because they inherit one mutant allele, which is a big predisposition to getting cancer
ex. retinoblastoma, those w/ the inherited mutant allele have 90% chance of developing a retinoblastoma tumor
What are 3 examples of external growth factors from which cancer can be stimulated?
- platelet-derived growth factor
- epidermal growth factor
- steroid hormones
-made in other cells
What are 3 kinases in signal transduction pathways that can be altered with cancer?
- Src tyrosine kinase
- mitogen-activated kinase (MAPK)
- Jun kinase (JunK)
What are 3 transcription factors that are often involved in cancer?
- MYC
- FOS
- JUN
What are the 2 main effects of cancer alterations to transcription factors?
- induce genes that promote cell divison
- repress genes that inhibit entry into cell cycle
4 places for regulation of cell growth
- growth factors
- growth factor receptors
- signal transduction molecules (tyrosine kinases)
- nuclear transcription factors
Explain the 2 hit model of carcinogenesis
= a cell can initiate a tumor only when it contains 2 damaged alleles
At least 2 mutations (hits) are required for a tumor suppressor to cause cancer. 1st can be germline (retinoblastoma) and the second hit is sporadic in embryo. This explains bilateral retinoblastoma in the inherited version.
If the first isn’t inherited, both mutations would need to occur somatically, which is why it’s unilateral for uninherited retinoblastoma.
What are 3 possible mechanisms for the retinoblastoma-susceptibility gene (RB1) hits?
- point mutation
- deletion
- hypermethylation
How does a loss of fxn mutation come about?
needs 2 hits
What is RB1?
=retinoblastoma 1 gene
-a tumor suppressor gene
What is a feature of tumor suppressor genes that is a contraindication?
Inherited mutations are dominant alleles at the level of the individual (ie. heterozygots usually develop the disease), but they are recessive alleles at the cellular levels (ie. heterozygous cells do not form tumors)
How is the contradiction of tumor suppressor genes explained?
Individuals who inherited the 1st hit, a second hit in any one cell will cause a tumor.
Incomplete penetrance of the retinoblastoma mutation (90%) is explained by some people with the 1st hit don’t experience a second hit
Which form of the RB1 protein is active?
When it is unphosphorylated
-it is downregulated when it’s phosphorylated by CDKs before the S phase
What happens when Rb is unphosphorylated?
It is active and pRb binds to the E2F transcription complex, inactivating it.
E2F is required for cell’s progression into S phase, so pRb binding halts the cell cycle
What can cause permanent inactivation of Rb1?
- A loss of fxn mutation
- deletion of the gene
- hypermethylation of its 5’ region
What 4 cell growth regulators are proto-oncogenes involved in regulating?
- growth factors
- growth factor receptors
- signal transduction molecules
- nuclear transcription factors
How does a proto-oncogene become an oncogene?
When a mutation occurs and causes it to be an excessively active, leading to unregulated cell growth and differentiation
When is a cell transformed?
When a cell proceeds from regulated to unregulated growth
When is a cell transformed?
When a cell proceeds from regulated to unregulated growth
How are oncogenes activated?
- by gain-of-fxn mutations or gene amplification
- hypomethylation of the oncogene’s 5’ region -> increased transcription
- chromosome rearrangements that up-regulate the oncogene
How are onocgene mutations different than tumor suppressor mutations?
oncogenes are typically dominant at cellular level, so only a single mutated copy is needed to cause tumorogenesis
How do inherited mutations differ between tumor suppressors and oncogenes?
Most tumor suppressor genes exhibit germline mutations that can cause inherited cancer syndromes
Germline oncogene mutations that cause inherited cancer syndromes are uncommon
TS & Oncogene Summary
What are 3 methods used to ID specific oncogenes?
- retroviral definition
- transfection experiments
- mapping in tumors
What are 3 methods used to ID specific oncogenes?
- retroviral definition
- transfection experiments
- mapping in tumors
What is a retrovirus?
A type of RNA virus that can use reverse transcriptase to transcribe its RNA into DNA, which then gets inserted into a chromosome of a host cell
-some retroviruses carry mutations of growth-promoting genes into cells
What oncogenes have retrovirus studies helped us ID?
- epidermal growth factor (EGF) through the ERBB oncogene
- RAS (ra t s arcoma) - altered in ~25% of human cancers
Nuclear transcription factor genes:
1. MYC
2. JUN
3. FOS
What are 4 proto-oncogenes that germline mutations can give rise to?
- MET
- RET
- CDK4
- KIT
What are transfection experiments?
Material from human tumor cells are transferred to nontumor cells -> transformation of recepient cells
-RAS found to occur naturally as proto-oncogene in human genome
What is tumor mapping?
Observing chromosomal rearrangements, like translocations (BCR-ABL in CML or t(15;17) fusion of RARA and PML in APL)
What is genomic instability?
Mutations, chromosome breaks, aneuploidy
-all contribute to tumorigenesis b/c they can activate oncogenes or deactivate tumor suppressor genes
-also assocaited w/ hypomethylation of DNA (common feature of tumors)
How is telomerase effected in cancer?
It can get activated to escape cellular senescence
What is the normal role of telomeres?
On the tips of chromosomes, which cannot be replicated by DNA polymerase
-once it’s reduced to a critical length, a signal causes the cell to become senescent or undergo apoptosis, limiting proliferation
-tumor cells overcome this by activating the gene that encodes telomerase
What is telomerase?
A reverse transcriptase that replaces telomeric segments normally lost during cell division
-rarely present in norm cell, but in 85-90% of tumor cells
-> uninhibited growth w/ accumulation of mutations
-somatic mutations in promotor region of TERT gene, in 70% melanomas
What are 3 approaches to IDing tumor suppressor genes?
- linkage analysis w/ polymorphic markers to ID the chromosomal segment w/ a cancer-causing mutation
- observing a specific chromosome segement deletion (by viewing SNPs or STRs) in tumor cells (as the 2nd hit) gives a map location for the inherited mutation (1st hit)
- genome-wide associated studies (GWAS) - compares thousands to millions of SNPs assayed on microarray
What lab methods can be used to detect tumor suppressor deleted locations on chromosomes?
- CGH
- SNPs
- whole-genome sequencing
-after loss of heterozygosity found, the DNA sequences can be tested to ID tumor-causing mutations
What are STRs?
microsatellites
segment tandem repeats
What are 2 ways epigenetic changes can be analyzed?
- methylation patterns
- histone modification
What is epigenetics?
The study of changes in gene expression or phenotype caused by mechanisms other than variation in DNA sequences
-methylation, histone modification, mRNA-binding by microRNAs
What 2 actions can cause chromatin condensation?
- methylation of gene’s promotor region
- histone hypoacetylation
What does chromatin condensation lead to?
inhibits binding of transcription factors to a gene’s promotor -> reduced gene expression
Can epigenetic changes be reversed?
Yes, with drugs
-demethylating agents
-histone deacetylase (HDAC) inhibtiors counteract histone hypoacetylation that can silence tumor suppressor gene activity
What is a driver gene?
A gene that contributres directly to a growth advantage in tumors and are primary causes of cancer
-ex RB1 gene
What is a passenger gene?
genes that undergo somatic mutations during tumorigenesis but don’t directly confer growth advantages to cells
What is the NF1 gene?
=neurofibromatosis type 1 gene
-gene product = neurofibromin
- associated w/ GAP (GTPase-activating protein)
- neurofibromin down-regulates RAS
What is the most commonly altered cancer-causing gene?
TP53 gene
-alterations in many CRC, breast, lung
What effect do TP53 mutations have?
Dominant negative, so the 2-hit model does not apply
What does the TP53 gene product do?
=p53
-increases in response to cell damage, acts as a transcription factor to help regulate dozens of genes that affect cell growth, proliferation and survival
What happens when TP53 is mutated?
Cells w/ damaged DNA can evade repair and destruction
What is the role of TP53 in Li-Fraumeni syndrome?
-inherited as a germline mutations
-transmitted as autosomal dominant
What is a second mutated tumor suppressor gene that contributes to LFS?
CHEK2
-encodes a kinase that normally phosphorylates p53 as a response to ionizing radiation
-loss of fxn -> lack of p53 activation
What is FAP?
=familial adenomatous polyposis
-many colonic adenomas in 2nd or 3rd decade of life (colonic adenomas are the immediate precursors to colorectal cancer)
-autosomal dominant subtype
What is the mortality rate of CRC in the US?
1/3
What is the most common APC mutation?
nonsense or frameshift -> truncated protein product
-assocated w/ CRC
-germline mutations are rare
-somatic mutations in 85% of CRCs
What is the test for an inherited APC mutation?
Protein truncation test
-more often rely on DNA-based tests
What is APC?
a tumor suppressor gene
-both copies of APC must be inactivated for tumor progression
-in 85% of all noninherited CRCs
What are 3 ways APC protein acts as a tumor suppressor?
- phosphorylation and degradation of beta-catenin in Wnt signal transduction pathway (dampens signal that -> cell prolif)
- involved in cell-to-cell (or matrix) adhesion properties (beta-catenin and E-cadherin)
- expressed in microtubules that pull chromosomes apart during meiosis, so can -> altered microtubule activity
What is Lynch Syndrome?
AKA hereditary nonpolyposis colorectal cancer (HNPCC)
-autosomal dominant, high-penetrance
-40-60% caused by mutations in MSH2 gene
-25-30% in MLH1 gene
-lots of microsatellite loci instability -> lack of DNA repair
What 2 genes are major contributors to breast cancer?
BRCA1 and BRCA2
What is different about BCRA1/2 mutations compared to RB1 and APC?
All inherited mutations cause a loss of function, then a second somatic mutation -> cancer.
-In contrast to RB1 and APC, somatic mutations affecting BCRA1/2 are seldom seen in sporadic (noninherited) breast tumors
What are the roles of BRCA1 and BRCA2?
- protein product of BRCA1 is phosphorylated and activated by ATM and CHEK2 kinases in response to DNA damage
- BCRA1 protein product binds BRCA2 product, which then binds RAD51, a protein involved in ds DNA breaks
- their inactivation results in incorrect DNA repair and genomic instability
- other TS activity via interactions w/ p53, pRb and Myc by helping maintain genomic stability
What causes familial melanoma?
Can be caused by loss of fxn mutations in CDKN2A tumor suppressor gene
OR
by gain of fxn mutations in CDK4 proto-oncogene
-both -> loss of cycle control via pRb and p53 pathways
What mutations are seen in most sporadic melanomas?
CDKN2A
BRAF
and/or
NRAS
What is the germline mutation with a significant contribution to prostate cancer?
HOXB12
What somatic muations contribute to prostate cancer?
mutations in the androgen receptor gene and additional DNA repair genes
What mutation -> Hirschsprung disease?
Loss-of-fxn mutations in RET proto-oncogene
What can germline gain-of-fxn mutations in RET cause?
3 types of inherited, autosomal dominant multiple endocrine neoplasia type 2 (MEN2):
1. MEN2A
2. MEN2B
3. a syndrome w/ familial MTC
What is the role of RET?
a proto-oncogene that encodes a receptor tyrosine kinase
-involved in embryonic neural crest cell migration
-activated by complex of GDNF and GFRalpha
-interacts w/ RAS pathway
What determines the size of a PCR product?
Distance b/w primers
What does microsatellite instability look like on PCR?
- If there’s expansion, the repeats will be higher up/heavier
- Contraction -> lower/distal
- mixed effects -> lower/distal and higher/heavier. Or spreading out across a large area showing greater distribution
