DNA Repair & Cancer

genetic / genomic testing for breast cancer

1. Direct testing for BRCA mutations 2. Testing for HER2 amplification (HER+) breast cancer 3. Multivariate Testing to get a statistical likelihood (prediction) of how the cancer will behave based on genomic data - Response to chemotherapy, Prediction for metastasis eg. MammaPrint, Oncotype DX

Oncogenesis by Gene Amplification

1. Double minutes 2. Homogeneously Staining Regions

Familial/Inherited/Mendelian vrs Non-familial/Sporadic Cancers Caused by Tumor Suppressor Gene mutations

1. Familial = 10,000 x incidence of sporadic (penetrance is roughly 90% for familial) 2. Familial usually has multiple tumors 3. Familial usually has earlier onset (since in familial 1st germline mutation is inherited while in sporadic, both genes are normal at birth) In both Familial and sporadic only a small portion of cells become cancers

Cancer Causing Genes

1. Oncogenes from proto-oncogenes that normally stimulate growth . GAIN OF FUNCTION 2. Tumor suppressor genes - normally inhibit growth. LOSS OF FUNCTION 3. Repair genes - normally limit frequency of mutations

Oncogenic Activation Mechanisms

1. Point mutation 2. Truncation/deletion 3. Translocation (Illegitimate Recombination)

Serious Mutagens

1. UV (from sun or tanning beds) 2. Ionizing radiation (from space or the ground) 3. Aflatoxin (from moldy grain) 4. Benzene 5. Formaldehyde 6. Mustard gases

Mechanisms producing 2nd hit

2nd hit is usually caused by aberrant/inappropriate methylation.

Double Stranded Breaks

A difficult type of mutation to repair which is particularly dangerous to dividing cells (high probability of loss of genetic material). Two repair mechanisms: 1. Non-homologous end joining - more common method; does not use homologous chromosome to repair the break 2. Recombinational repair - does use homologous chromosome; less error prone than NHEJ

Ras

A proto-oncogene is a cytosolic signal transducer with GTPase involved in the MAP Kinase Mitogenic Signalling pathway. A point mutation at Gly12 or Gln61 makes Ras constitutively active (Ras is active even in the absence of a growth factor) and cell grows unregulated. Ras is a common mutation in most cancers.

de la Chapelle Syndrome/Atelosteogenesis Type II

A severe Autosomal Recessive disorder of cartilage and bone development. Infants born with this condition have very short arms and legs, a narrow chest, and a prominent, rounded abdomen, cleft palate, distinctive facial features, clubfoot and hitchhiker thumbs. Caused by mutations in the SLC26A2 gene. This gene provides instructions for making a protein that is essential for the normal development of cartilage and for its conversion to bone.

Tautomerism

Ability of certain chemicals to exist as a mixture of two interconvertible isomers

As part of a chemotherapy protocol, a 59-year-old female patient is administered the DNA damaging agent cisplatin with the hope that apoptosis will be induced in the cancerous tissue. Which of the following occurs to facilitate this programmed cell death response in the patient?

Activation of pro-caspase 3 by proteolytic cleavage

Rubinstein-Taybi Syndrome

Autosomal Dominant. Characterized by short stature, moderate to severe intellectual disability, distinctive facial features, and broad thumbs and first toes. Mutations in the CREBBP gene which provides instructions for making a protein that helps control the activity of many other genes. EP300 gene cause a small percentage of cases. It appears to be important for development before and after birth.

Bloom Syndrome

Autosomal Recessive, DNA Repair Defect caused by mutations in the BLM gene. BLM has DNA HELICASE activity which is required for recombination and replication repair. Symptoms: stunted growth, facial rash with UV exposure, chromosomal instability. Chromosomal instability results in many breaks and sister chromatid exchanges which increases risk for a broad range of cancer types. Both males and females have low genetic fitness but males of 0 genetic fitness.

Maple Syrup Urine Disease

Autosomal Recessive. Body is unable to process certain protein building blocks (amino acids) properly. Mutations in the BCKDHA, BCKDHB, and DBT genes can cause maple syrup urine disease. Mutations in any of these three genes reduce or eliminate the function of the protein complex, preventing the normal breakdown of leucine, isoleucine, and valine.

Breast and ovarian cancer

BRCA1 and BRCA2 is for repair of ds DNA breaks by homologous recombination and apoptosis. Women BRCA1 or BRCA2 single gene mutations (locus heterogeneity) have up to an 85% risk of developing breast cancer by age 70 and the risk of developing ovarian cancer is about 55% for BRCA1 and 25% for BRCA2 mutations. BRCA1 gene mutations show allelic heterogeneity.

Cancer

Cancers are manifestations of usually somatic errors. Predisposition to cancer can be inherited in a Mendelian fashion, but may exhibit non-Mendelian family clustering suggesting multifactorial causation. The threshold model does not apply for cancer. Rather, it is viewed as a disease that progresses by the accumulation of genetic alterations. The property of progressive aggressiveness is unique to cancer. Tumour progression results from waves of mutation followed by clonal expansion.

Are cancers derived from a single cell or many cells?

Cancers are usually monoclonal. Proven by: 1. X-inactivation pattern (same x is inactivated in every cancer cell) 2. Chromosomal abnormalities/aberrations are contained in every cell in the tumour 3. Multiple myelomas produce a monoclonal Ig (same antibody).

Spontaneous lesions

Changes that occur in a resting cell due to the chemical nature of the DNA. Increased by exposure to mutagens such as sunlight, X-rays, BBQ chicken, bananas. Three main types: 1. Depurination 2. Deamination 3. Oxidative damage

Some people believe that astronauts are at a greater risk for ageing diseases and cancer due to gamma γ-rays (gamma-rays) and cosmic rays found in outer space. Without the protective effect of the Earth's magnetic field and atmosphere, this ionizing radiation can strike a person and cause DNA damage. What type of damage are astronauts most at risk for due to this ionizing radiation?

Chromosomal breakage. Ionizing radiation such as X-rays, gamma rays, and cosmic rays have such high energy that collision of this ionizing radiation with DNA tends to cause a strand break. Thymidine dimers form by interaction with ionizing radiation such as UV light. Apurinic sites spontaneously form, and mismatched bases spontaneously may be found after DNA synthesis.

α-thalessemia

Chromosome 16 contains the α-cluster. Because of the repetitive structure of the α-cluster, deletions are the common disease causing mechanism for α-thalessemia.

Common Inherited Cancers

Colorectal 1. Familial Adenomatous Polyposis (FAP) 2. Hereditary Non-Polyposis Colon Cancer (HNPCC): Lynch syndrome Breast/Ovarian

DNA Damage

DNA Damage is constantly occurring due to replication errors and external factors like UV light, chemicals, radiation, pH, smoking. Cancer and ageing are the long term consequences of DNA Damage. Cancer develops when cells with damaged DNA replicate.

Xeroderma Pigmentosum (XP)

DNA Repair Defect that causes mutations in 9 XP genes (locus heterogeneity). Affects nucleotide excision-repair and causes thymine dimer formation. Higher incidence in Japan than Europe-USA. Leads to extreme sun sensitivity (sunburn, blistering, freckles), skin cancer including melanomas, ocular tumors and conjunctivitis. DNA damage is cumulative and irreversible.

DNA Damage vrs DNA mutation

DNA damage is constantly occurring however it can be recognised and efficiently repaired. Trying to replicate through it can convert DNA damage into mutations.

Role of Epigenetics in Cancer

DNA methylation and histone modifications are frequently altered in tumor cells. 1) Silencing of tumor suppressor loci causing cell overgrowth 2) Loss of imprinting causing activation of growth associated genes (e.g. IGF2) methylation loss causes creation of oncogenes. 3) MicroRNAs - miRNAs act to reduce the expression of genes by targeting specific mRNAs. miRNAs have been shown to undergo changes in expression in cancer cells with frequent amplifications and deletions of miRNA loci

DNA mutation

DNA mutation is spontaneous or induced, however induced mutations simply increase the rate of spontaneous mutations.

Retinoblastoma

De novo mutation which cannot be inherited or Autosomal Dominant due to germline. Mutations in the tumor suppressor RB gene on chromosome 13, which helps regulate G1/S phase transition, lead to childhood retinoblastoma and osteosarcoma. RB- (mutant) cannot bind E2F. Virtually all cancer cells show dysregulation of the G1-S checkpoint as a result of mutation in one of four genes that regulate the phosphorylation of RB: RB, CDK4, cyclin D gene, and CDKN2A (p16). Inherited retinoblastoma is bilateral, early onset and multiple tumors. Sporadic retinoblastoma is unilateral, later onset and single tumor.

Translocation (Illegitimate Recombination)

Exchange of information with non- homologous chromosome. This can cause a mutation in photo-oncogenes and lead to disease eg. Burkitt Lymphoma (activation of myc), Chronic Myeloid Leukemia (activation of abl)

Double minutes

Extrachromosomal fragments of DNA containing an amplified region of the chromosome. Often seen in tumors where the amplified region includes an oncogene e.g. EGFR oncogene is often amplified as double minute chromosomes in advanced gliomas. Visualised with FISH probes.

oncologist thought it would be a good idea to test if a biopsy of new cancer foci were HER+. Which of the following diagnostic tests would most be able to identify this type of genomic aberration in this patient?

FISH

Sporadic Breast Cancer

HER-2 (Human Epidermal growth factor Receptor 2) overexpression in sporadic breast cancer. HER-2 amplification forms double minute chromosomes. HERCEPTIN (antibody) binds to HER2 and prevents binding of EGF to HER2. Herceptin is very effective for Her2+ tumors but not effective for treatment of Her2- tumors.

Homogenously staining regions (HSRs)

HSRs are areas of chromosomes in cancers that contain amplified oncogenes e.g. N-MYC amplification in neuroblastomas (NB). HSRs are due to chimeric fusion. HSRs = oncogenic amplification.

Microarray Analysis

Helps in cancer classification. Accuracy of typing is critical to design appropriate therapy. Requires molecular determination of genes being expressed in order to determine: • rate of proliferation • capacity for invasion • potential of metastases

Strand Discrimination

How does the cell knows which strand is not mutated. It appears to be due to interaction with replication machinery and also involves methylation.

Ionizing Radiation

Include cosmic rays, γ-rays, X-rays, UV rays, radioactive particles (α, β, γ) and terrestrial radiation. Causes extensive damage to DNA (base damaging type) including HERITABLE MUTATIONS. Also cause pyrimidine dimers.

Nucleotide Excision Repair (NER) Mechanism

Indirect Repair Mechanism which removes up to 30 bases around a damaged site. This is how pyrimidine dimers formed by UV damage are repaired. Specific repair proteins recognise damage and remove the damaged base or region around the damaged base then shared repair proteins replace the excised region.

Base Excision Repair/Short Patch Repair

Indirect Repair Mechanism which repairs a single (or just a few) damaged bases by removing it (damage by methylation, oxidation, etc.). Damaged nucleotides can be removed by DNA glycosylases which recognize specific damaged bases in DNA, break the N-glycosidic bond, remove the sugar-phosphate by endonuclease, replace the base and ligate the DNA. There are many different types of DNA glycosylases which are specific for different aberrant bases e.g. uracil glycosolase.

Mismatch Repair(MMR) / Post Replication Repair / Mutator genes

Indirect Repair Mechanism which repairs mismatched bases that form due to tautomerism. Also very important in relation to removing small repeats that tend to expand e.g. triplet expansion disorders. Associated with colon cancer.

UV light

Interfere with normal pairing and block replication. Create pyrimidine dimers, or thymine dimers.

Deamination

Loss of amine group from base particularly cytosine. Cytosine deaminates to form uracil which likes to pair with A. This happens relatively frequently but it is easy to fix since U does not belong in DNA. However 5-methyl cytosine deaminates to thymidine. This deamination creates a MUTATIONAL HOTSPOT.

A cell is exposed to excessive ionizing radiation which results in severe damage to its biomolecules. Due to this damage, apoptosis is initiated. Which organelle becomes permeable as part of the initiation of this programmed cell death process?

Mitochondria

Depurination

Most common form of spontaneous lesion caused by the removal of a purine. Glycosidic bond between base and sugar in purine nucleotides is broken. Sugar-phosphate backbone remains but base is lost creating an APURINIC (AP) SITE. If it persists through replication then mutation can occur

Oncogenes

Mutant or misregulated proto-oncogenes which are: 1. Growth factors 2. Growth factor receptor tyrosine kinases 3. Cytoplasmic tyrosine kinases 4. MAP Kinase Pathway proteins and receptors (Myc) 5. Proteins with GTPase activity 6. DNA-binding nuclear protein transcription factors These proto-oncogenes are involved in cellular growth control, so when mutated, they are CONSTITUTIVELY ACTIVE. There is production of large amounts of proteins that stimulate cell division and therefore unlimited cell proliferation. Only one porto-oncogene needs to be mutated in order to develop cancer.

Familial Adenomatous Polyposis (FAP)

Mutation in APC tumor suppressor gene on chromosome 5 leads to bowel carcinoma. APC is part of the WNT signally pathway which normally prevents β-catenin degradation which activates growth genes. Multiple (>100) adenomatous polyps develop throughout distal colon. Cancer is slowly progressive/cumulative. FAP has very high penetrance and Allelic Heterogeneity.

WAGR Syndrome/Wilms tumor, aniridia, genitourinary anomalies, and mental retardation syndrome

Mutation in WT1 tumor suppressor gene leads to Wilm's tumor. Most people with WAGR syndrome have aniridia, an absence of the iris. Usually early onset so children presents with tutors in many organs particularly kidneys. Caused by a deletion of genetic material on the short (p) arm of chromosome 11. Disease is not inherited.

Hereditary Nonpolyposis Colon Cancer (HNPCC)/Lynch Syndrome

Mutation of DNA mismatch repair (MMR) genes/mutator genes. Locus heterogeneity: MSH2 (60%), MLH1 (30-35%), PMS1, PMS2, or MSH6. Cells accumulate mutations at rates up to 1000 times higher than normal resulting in microsatellite instability (STR/SSR show size variability due to inaccurate replication). Unlike FAP, have few polyps instead of >100 and cancer progresses rapidly.

Burkitt lymphoma

Myc (a nuclear transcription factor important for G1/S transition) proto-oncogene is translocated from chromosome 8 to chromosome 14 where it is fused to immunoglobulin locus. Myc oncogene expression is increased under the very active IgH promoter regulation. Lymphocytes fail to differentiate (undergo rapid cell divisions) which leads to cancer.

Common Mutagens

Not as dangerous as the Serious Mutagens but considered because of their impact. 1. Alcohol (acetaldehyde breakdown product) 2. Barbecued hamburgers 3. Cigarettes

Loss of Heterozygosity (LOH)

Parents are homozygous at a locus (allele 1 or 2), child is heterozygous but the tumor tissue has only a single allele type (allele 2) which causes 2nd 'hit' and tumor initiation.

Transversion mutation

Purine to pyrimidine mutation or vice versa

Which of the following disorders may be described with the liability threshold model?

Pyloric stenosis. Boys have a lower threshold or a greater likelihood of having the disorder. Therefore when a girl has pyloric stenosis, the chance of future siblings having the disease is higher than for a male since it means the parents together have more bad genes than the normal population.

Which of the following activities best describes the function of the NF1 protein?

RAS GAP (inhibits RAS activity) E.

Li-Fraumeni Syndrome

Rare Autosomal Dominant. Inherited p53 tumor suppressor gene mutation greatly increases the risk of cancer at a young age in many different organs. Can result in several kinds of cancer including breast, bone, brain, adrenocortical tumors and soft-tissue carcinomas bladder

Ataxia-telangiectasia (AT)/Louis-Bar

Rare Autosomal Recessive, DNA Repair Defect caused by mutation in ATM gene. ATM is a ser-thr kinase that detects DNA damage, activates cell cycle arrest and DNA repair proteins, and repairs dsDNA breaks. AT affects cerebellum (ataxia) and immune system, increases incidence of cancer and ocular telangiectasia common. Also have cellular gamma-ray sensitivity.

Fanconi Anemia

Rare Autosomal Recessive, DNA Repair Defect. Locus heterogeneity however most cases are due to mutations in FANCA, FANCC, and FANCG genes. These genes code for proteins involved in DNA interstrand cross-link repair. Mutation causes increased spontaneous chromosome breakage which is made worse by exposure to DNA cross linking agents. Leads to radial ray defect, pancytopenia (low cell production), short stature, leukaemia, and increased risk of neoplasia.

Oxidative damage

Reactive oxidative compounds e.g. superoxides, peroxides cause oxidative damage to many parts of cell including addition of oxygen groups to nucleotide bases e.g. 8-oxo-7-hydroxyguanosine. Results in mis-pairing with A and potential transversion.

One mechanism that p53 employs to promote apoptosis is to induce the synthesis and secretion of IGFBP3 (Insulin-like Growth Factor Binding Protein). What occurs as a consequence of this p53 function?

Reduction in tyrosine kinase activity

Somatic Errors vrs Germline Errors

Somatic errors lead to cancer and ageing but are not inheritable while Germline error result in inheritable genetic diseases. Both errors, however, allow for evolution.

An individual prostate cell undergoes apoptosis in response to activation of the Perforin / Granzyme Pathway. Of the choices listed below, which was the earliest event that contributed to this cellular decision?

Stimulation by a cytotoxic T-Cell

Frameshift mutations

Tend to occur at positions where there are base repeats e.g. GTCGAAAAACTCA because of 'slipping' of DNA polymerases during replication of these repeats. DNA loops or kinks at these points causing expansion or contraction of these repetitive sequences. This explains Huntington's and Fragile X syndrome The repetitive regions can be highly polymorphic.

Mutagens

Things that increase the frequency of 'normal' mutations eg. mismatches, depurination

DNA Proof-reading

This is a DNA repair mechanism. DNA Poly has 3' to 5' exonuclease activity. This DNA repair mechanism occurs in S Phase.

Chronic Myeloid Leukemia

Translocation of abl gene from chromosome 9 to chromosome 22. Hybrid bcr-abl fusion protein makes cytosolic tyrosine kinase CONSTITUTIVELY ACTIVE. This mutation is somatic and non-heritable. The hybrid brc-abl fusion protein is competitively inhibited by GLEEVEC/Imatinib mesylate which is specific for abl tyrosine kinase.

p53 ("guardian of the genome")

Tumor suppressor at G1/S checkpoint that controls both cell birth and cell death. It slows cell cycle to allow time to do repairs, increases DNA repair capabilities and initiates apoptosis when damage is too severe. Loss of p53 function results in net cell growth, but also increased mutation frequency, which drives cancer progression. Mutated in more than 50% of all cancers.

Tumor Suppressor Gene

Tumor suppressor gene mutations lead to loss-of-function and then cancer. 'Two hits' or two mutations are needed for tumor initiation. Associated with a single susceptibility gene: 1. Inherited Autosomal Dominant Cancer Syndromes (actually recessive at the cellular level, but with guaranteed loss of second copy) eg. Retinoblastoma 2. Inherited Autosomal Recessive Cancer Syndromes of Defective DNA Repair eg XP, AT, Bloom syndrome, Fanconi anemia. Multifactorial causes: 1. Familial cancers e.g. breast cancer, ovarian cancer, pancreatic cancer, colon cancer

Spontaneous mutation

Two types: 1. Errors of replication - mistakes during replication e.g. wrong base is incorporated by DNA polymerase. Only occurs during S phase of cell division. 2. Spontaneous lesions - chemical changes that occur spontaneously. Occurs in G0 or resting cell.

DNA Repair Genes

Vulnerable to mutation which leads to: 1. Increased error rate - particularly true for proofreading and mismatch repair (MMR), produces mutator phenotype 2. Genomic Instability e.g. Bloom syndrome DNA Repair Defects lead to developmental delay, neurocognitive issues, extreme sensitivity to DNA damaging agents and increased risk of cancer.

Steroid Sulfatase Deficiency

X-linked Disorder with mutation on the STS gene.

A biopsy is taken from a patient with colorectal cancer. The gene expression profile of two thousand genes from this tumour isolate is compared to gene expression in nearby normal tissue. What technique allows for this comparison?

cDNA microarray

A biopsy is taken from an eight-year-old boy with a heritable disorder and some of his cells are used for research. His cells are studied in culture, and a mutation in a gene which encodes a DNA-helicase is discovered. What is the best description of the DNA damage that is likely to be observed in this patient?

chromosomal instability

Fathers who come down with HD are more likely to have children that exhibit anticipation of the disorder. Which term best explains a mechanism that accounts for this observation?

meiotic drive.

Transition mutation

purine to purine or pyrimidine to pyrimidine mutation