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Genetics ReviewI. MutationsA. Three main types:1) Substitutions: i) transitions = purine purine or pyramidine pyramidine and are harder to detect; transversersions = purine pyramidine or vise versa)a) Missense: a.a. substituted for anotherb) nonsense: stop codonc) silent or synonymous don’t alter a.a. but may destabilize mRNA or decrease rate of translation due to codon bias (less tRNA around for that codon)2) Insertions or deletions may be a single base or 1,000s; cause frameshifta) Transposons are large segments that can be insertedB. Chemical mutgens: each has its own spectrum, so it can only be reverted by similar mutagen1) Base analogues: look similar to base, but bind to wrong base (Ex. 5-bromouracil looks like T but can bind to G)2) Deaminating agents: remove peripheral amine group mispairing; Ex. Nitrous acid converts A hypoxanthine (binds to C); C U can also occur3) Alkylating agents: add methyl or ethyl group to base depurination (entire base deleted) or mispairing; Ex. EES and nitrosoguanidine4) Intercalating agents: planar aromatic compounds that insert in b/w bases stabilizes frameshift and mispairing; Ex. Acridine orange and proflavinC. Other mutagens1) Radiation causes breaks in DNA (direct damage) or it creates radicals (indirect) mispairings during repair = mutations2) UV pyrmidine dimers (mostly T-T; sometimes T-C) block DNA replication and activate repair process which makes many errors3) Spontaneous: have different spectrum (location and type); Ex. Deamination of 5-methyl cytosine thymidine (causes C/T “hotspots”)D. Studying mutants was possible after the discovery of inducible mutagenesis1) Direct Selection: only the mutant of interest will survive the restrictive condition (i.e. in presence of antibiotic)2) Indirect Selection: i) Prototrophs can divide in minimal medium (restrictive condition), but auxotrophs need a compound to divide; ii) Add penicillin blocks cell wall synthesis of dividing cells (prototrophs) death; iii) auxotrophs have been selected3) Screening via replica plating: i) Grow bacteria on plate, ii) transfer bacteria with velvet disc to new plates which have an indicator compound (Ex. EMB turns red when fermentation is happening) or lack a compound, iii) mutant colonies (colored or dead) identified on original plate due to conserved pattern on both platesa) Helpful for conditional mutants (ex. Temperature-sensitive) because they die in the restrictive condition (ex. Hotter temperature)E. Mutants can teach us about five things:1) Complementation: whether two mutants are in the same gene; Ex. Fusing genomes of two different XP patients if patient is no longer UV sensitive, mutations arose in two different genes2) Cellular process: temperature-sensitive mutants showed that some genes important in initiation while others important in actual replication; also ordered biochem. Pathway- Pleiotrophic: mutant that affects multiple processes3) Reversion: return to wild type gene sequence vs. Suppression: second mutation restores wild type phenotypea) Intragenic suppression: Ex. 1st mut. Makes protein temp-sensitive and 2nd mutation increases thermal stability; ex. Insertion frameshift suppressed by deletionb) Extragenic suppressionc) Translational suppression: nonsense suppressor tRNAs insert Tyr instead of STOP to lengthen prot.4) Genetic mapping: i) Take two viruses that grow only with suppressor tRNAs and test for complementation by adding them to cell w/o s-tRNAs; ii) If the mutations are on separate genes, then put them into a cell w/ s-tRNAs to grow progeny; iii) add progeny to host w/o suppressor tRNAs and # that survive now gives frequency of recombination (genetic distance)5) Test mutagenicity of compound via Ames Test: Add compound to His- strain (can’t grow w/o His), then see if the bacteria reverted to His+ (can grow w/o His)F. DNA Repair1) Bacteria have SOS response: genes involved in DNA repair activated with small amount of UV light (which cleaves LexA repressor prot.)2) Direct repair of Damagea) Pyrimidine dimers (by UV light) reversed by DNA photolyase and visible lightb) Ada (alkyltransferase) accepts alkyl group from P-group on damaged DNA activates its own transcription to also remove alkyl group from damaged Gc) Ligation of single-strand breaks by DNA ligase3) Excision repair (ER) using complementary strand as correct templatea) Base ER: Damaged base cut out leaving AP site APendonuclease recognizes AP site to nick the DNA dRPase cuts out the remaining nucleotide DNA pol. I and ligase repair the gap (Note: protons create thousands of AP sites per day constant repair)b) Nucleotide ER: thymine dimer (or aflotoxin) causes distortion in DNA recognized and uvrABC nuclease cuts out 12 bases; DNA pol. I and ligase repair gap - Defects here cause Xeroderma Pigmentosum - Transcription-Coupled Nucleotide ER allows cell to prioritize repair of active genes; Defect here causes Cocakyne Syndromec) Mismatch Repair: mut proteins patrol DNA looking for mismatches that were missed by DNA pol; once it finds mismatch, it cuts out new strand (recognized by methylation b/c methylase lags) - Defects cause hereditary nonpolyposis colorectal cancer (HNPCC)4) Recombination repair using homologous chromosome as correct template5) Trans-lesion bypass polymerases are low-fidelity and add any base to get past lesionII. Chromosomes: segments of genome that allow for variationIn meiosis I, maternal and paternal versions of each chromosome separate; in meiosis II, sister chromatide spearateSpermatogenesis takes 64 daysOogenesis pauses at prophase I; during ovluation, an oocyte completes meiosis I with one daughter cell becomes secondary oocyte and the other becomes polar body; meiosis II occurs only if fertilization happens, producing 2nd polar body and a mature ovumIn females, one of the two X’s is randomly inactivated early in development all progeny will be silent for those genes; seen as condensed chromatin (Barr body)Inactivation starts at Xic (X-inactivation center) and spreads along chromosomeXic contains gene that codes for Xist (only expressed in inactive X)Xist RNA coats entire inactive X chromosome; also inactvated by more cytosine methylation and less histone acetylationY chrom. has testis determining factor gene and signals spermatogenesisEnds are similar to X chrom = “pseudoautosomal” b/c these genes are diploidSRY: sex-determining region has very few genesAneuplodies usually arise from nondisjuction in first meiotic division because all four gametes have incorrect # of chrom.s (either double or none)More common in older mothers because meiosis is supsended at prophase IStructural rearrangements occur when chrom.s break and rejoin abnormallyTranslocation TypeChrom #MechanismClinical EffectBalancedSameReciprocal translocation b/w two nonhomologous Chrom.sPhen. Normal, but can pass down unbalanced translocationRobertsonian-1Two acroscentric chrom.s joined one metacentricOnce these translocated chrom.s undergo meiosis, 3 things can happen:Alternate segregation (good) = balanced gametesAdjacent 1 segregation (bad) = unbalanced but homologues separateAdjacent 2 segregation (worst): unbalanced and homologues pass to same cellCML is accompanied by Philadelphia chromosome (t9,22)Encodes more pumps to get ride of toxic materials = resistant to chemotherapyIII. Pedigree Analysis ComplicationsLocus heterogeneity: mutations in different genes cause same phenotypeEx. Retinitis pigmentosaNon-penetrance: Some individuals with mutation don’t show phenotypeEx. Penetrance is 80% for Van der Woude Syndrome (20% have non-pen.)Variable expressivitiy: Not all individuals show all features of the diseaseMosaicism: If child inerhits random germ cell mutation, chances are sibling will not unless mutation occurred early in parent’s development many gametes have mutationGenes on same chrom. can appear linked parent combo of traits > non-parental copyIntrachromosomal recombination (linkage) causes a higher frequency of parental gametes compared to recombinant gametes1 map unit (centimorgan, cM) = recombinant frequency of 1% (about 1000kb)LOD = log(likelihood of data if loci are linked/likelihood if loci are unlinked)+3 or higher is definite evidence of linkage; -2 or lower is exclusionIV. Genetic TechnologiesRestriction enzymes: cut across both strands of DNA at specific sequences leaves “sticky” ends which can be joined together by DNA ligasePlasmids: Use restriction enz. to cut vector (w/ antibiotic resistance gene and origin of DNA replication) open and insert human (or other foreign) DNA; insert plasmid into bacteria and grow on plate w/ antibioitic leaves only identical plasmidsAllows you to create libraries of cDNA and genomic DNAComplimentry DNA produced by starting with mRNA (has poly-A tail for stability and allows recognition by poly-T tail); reverse transcriptase makes cDNA strand; remove RNA and make second DNA strand; cloned into vectorcDNA more stable than mRNA; allows determination of introns/exonsHybridization: dsDNA denatured by heating or high pH (adds charge to P groups)Target is immobilized and probe (radiolabeled) added in solutionProbe may also be detected enzymatically; ex. add biotin to DNA binds to avidin which is cross-linked to horse radish peroxidase (produces colored product)Fluorescent tag may also be added; may have different colors for diff. probesCompare ratio of red:green for two different sequencesSynthesizing DNA<100bp: Growing chain anchored to resin; free nucleotide has labile protecting group to insure only one nucleotide added per cycle; PG removed before adding nucleotides; excess material washed away at each cyclePCR: amplifying a specific sequence of DNA; starts with 2 primersSequencing DNASanger method: Add DNA pol, 4 nucleotides, and a small amount of 4 fluorescent nucleotides (diff. colors) with chain terminating inhibitorsGel electrophoresis separates chains by size line them up to determine sequenceNexGen using emulsion PCR (separate aqueous drops are stable and each one contains only one starting DNA template molecule) and bridge PCR:Add adapters to ends of target DNA; separate two strands of DNA and add to glass slide (primed with complementary adapters linked on 5’ end); add nucleotides to synthesize new strand; heat and wash; cooling allows strand to find another primer new strand synthesized, heated, cooled, repeatTo “forest,” add 4 fluorescently labeled nucleotides (acts as chain inhibitor)Polymerize wash image deprotect; repeatIt’s also possible to measure light emitted when ATP is hydrolyzed by luciferase; ATP is made when Ppi released by DNA synthesisTissue culture: need thin tissue slices or dispersed cells to increase surface areaNeed antibiotics to keep fast-growing bacteria away from cellsHelps ID biochemical basis of disease; cell function; transfection (introducing DNA into cells); used as factories to make proteinsMonoclonal antibodies: inject mice with antigen mice makes antibodies in B-cells; remove B cells and fuse with tumor cells heterokaryon produces antibodies and grows forever grow cells and test for antibodies amplifyTransgenic mice: inject cloned, naked DNA into pronucleus after fertilization; implant fertilized egg into foster moth; analyze pups by PCR of tail DNAKnockout Mice: Clone DNA on both sides of target gene; Make targeting plasmid with long flanking arms that are complementary to the sequences on either side of the target gene (goal is to stimulate homologous recomb.); add drug-resistant gene in b/w flanks and lethal gene (ex. thymidine kinase which is sensitive to anti-viral drugs) outside of flanksNext, add plasmid to embryonic stem cell (brown fur) via electroporation; selection by adding antibiotic (or other drug) to kill cells w/o resistance AND antiviral drug to kill those that express TK (ensures homologous recomb. Into ES cell’s genome)Add selected ES cells to foster mother’s blastocyst (albino) chimeric pups are breeded with an albino mouse dark pups are 50/50 knockout mice (do PCR to verify)Tissue-specific knockout with Cre and Flp; tissue-specific promoters control these genes and when activated, Cre/flp (recombinases) recognize large sequences of DNA to cut DNA at those positions and re-join the strandsDelayed knockout: CreER(T) (fusion b/w Cre and ligand binding domain of estrogen receptor that doesn’t bind to estrogen but tamoxifen) is activated by tamoxifen migrates to nucleus and catalyzes DNA recombinationTiming of gene expression also accomplished by making tet repressor protein only bind to DNA in presence of antibiotics activates (doesn’t repress) transcription only genes which tet repressor has been engineered to recognize are activatedHuman gene therapy is more difficult than we thought initially: viruses (self-inactivating) can introduce DNA into cells and promote its stable integrationDNA expression decreased, viral cells were eliminated, and leukemia developed b/c virus integrated next to an oncogeneVaccines can now be creted by deleting genes that revert virus back to pathogenic stateStem cell therapy is hard b/c we don’t know what causes differentation hard to replicateV. The Human GenomeA. Gene # is about the same over species (22,000) but genome varies widelya. Heterochromatin (dark bands) are gene poor whereas euchromatin (light bands) are gene rich- Chrom 19 most gene rich major problems if this is messed up- 13, 18, and 21 gene poor viable trisomiesb. Gene size is highly variable (average = 30kb; some >2 Mb)c. cNCS (Conserved non-coding sequence): 5% of human genome is more conserved than we would expect functionally important b/c mut.s here would be selected outd. Regulatory elements Ided by chromatin immunoprecipitation (ChIP)i) short binding sites on DNA for transcription factorsii) May be close to promoter or distant (ex. Sonic Hedgehog enhancer)iii) Combined with NexGen sequencing to create maps of these regulatory seq.sRepetitive elements (45% of genome)Tandemly arrayed: Short tandem repeats (microsatellites), telomeres, centromeresInterspersed (molecular fossils): pseudogenes (mRNAs converted back to cDNA), mobile elements (SINEs/Alu, LINEs, transposons)Contribute to genome evolution and disease (via mutagenesis, recombination, and gene expression)Segmental duplications: evolutionary hotspots/chrom. instabilityHumans 99.9% the same; variation from:In/Dels (10%), STRPs/microsatellites (1-4 bp long; polymorphic) SNPs: frequencies and associations for 3 continents known; single bp variants; 3 million SNP differences b/w two peopleHigh Minor Allele Frequency (MAF) = early originHaplotype/LD blocks are highly conserved regions with same SNPs and low probability of recomb.Can use one SNP to ID surrounding SNPsHaplotype is like allele for an LD Block; can’t predict disease b/c plenty of healthy people have same haplotype but it can MAP the disease (ex. which genes are affected)CNVs: duplicated or deleted (more common); inherited or de novo; > 1kbIndividual may have 0-3+ copiesAlter risk of disease/gene expression by dosage effectTranscriptome (mRNAs vary at stage of development, disease state, etc.) and proteome depends on alternative splicing and post-translational processingIding sequence blocks conserved across species allows us to understand moreMice and humans have 217 conserved synteny (genes on same chrom/piece of DNA) blocks which includes 90% of respective genome1.5% of sequence encodes prot. (highly conserved)Homologs = genes from the same ancestor; orthologs = equivalent genes in two different species; paralogs = duplicated genes in same species Comparing us to neanderthals reveals changes in prot. Sequences and non-coding regions; strong selection for metabolism, cognition, and miRNAsGenomic DisordersUnstable regions in genome can be rearranged b/c of nonallelic homologous recombination (aka low copy repeats)Ex. DiGeorge /Velocardiofacial syndrome and microdeletion sndrome (developmental delay, heart disease, schizophrenia)VI. Complex Traits: Polygenic/multifactorial traits with a stronger environmental roleAppear later in life and are more common than monogenic disordersEx. Type 2 Diabetes: higher MZ vs. DZ concordance; sibling risk = 3.5>40 regions of genome Ided as having variantsMonogenic disorders complex too (modifier genes affect severity, progression, treatment)Heritability (h2)= fraction of risk for a phenotype due to genetic variationDifficult to find genes contributing to complex traits b/c:Small contribution from any paritcular gene; susceptibility alleles (difficult to recognize b/c more likely to have only a modest effect)May be caused by 1, a few, or a large number of genesPhenotype is continuous, not dichotomous; diagnoses based on observationsGenetic variants are common common disease; or multiple rare variants that lead to the same phenotypeMany people with “diseased” variant are healthy/incomplete penetrance; others may have phenotype for other reasons (phenocopy)LD blocks identified can still have 50 genes (low resolution of genome)How do we find the genes that contribute to complex traits then?Epidemiology: twin studies (concordance rate b/w MZ and DZ twins), relative risk measures (for sibs and offspring), pedigree analysisTwin studies could involve environ. factors (raised apart or together); R = 1 is a perfect correlation and R = 0 is no correlationDefining disease more clearly to reduce heterogeneity (age at onset, severity, etc.)Focus on specific populations (Finns, Amish, etc.) to reduce heterogen.Linkage analysis: use pedigrees and compare segregation of mapped markers (STRs or SNPs) with a disease phenotype; marker is “linked” if it co-segregates with phenotype more often than by chance alone (LOD>3)Association studies: Don’t need to collect families; compare frequency of allele in a population of affected individuals vs. unaffected individualsPositive association could mean: a) marker is cause of phenotype (rare), b) marker is in LD with gene causing phenotype, or c) marker and phenotype are unrelated (population stratification) need to do 2nd studyNegative result could mean: a) marker not associated w/ phenotype, b) too much heterogeneity, or c) multiple rare alleles so you missed the LD blockGWAS (Genome-wide): Finding a “risk allele” only means that person is 1.2x more likely to get the diseaseTransmission-disequilibrium test (TDT): scores transmission of allele from parents to offspring; used in linkage or association studies; if allele increases susceptibility of disease (or is in LD block w/ susceptibility allele), then it will be transmitted >50%After you find the region of the genome, you can sequence candidate genes (function fits with pathophys.) or capture and sequence entire region with NexGen Seq.Databases of normal variation (HapMap/1000 Genomes) allow us to know if the variant has been identified in control populationsEx. Age-related macular degenerration: vision loss, multifcatorial causationAgnostic search—found CFH gene (part of immune system) to contribute riskMZ twin concordance is 100%; difficult to study though b/c slow progressive disorder and older adults have compounding disorders as wellLinkage analysis revealed several chrom. involved but LOD of only 2Genomic profiling: detecting multiple gene variants associated with risk of diseaseOne region shows tight association w/ complement factor CFHAnother region (susceptibility locus) has two haplotypes; function unclearVII. Non-Mendelian InheritanceAnticipation: phenotype becomes more severe (earlier onset or worse) w/ each generationCaused by unstable repeat expansion mutations: normal gene has short nucleotide repeates (triplets); # of repeats is variable and can increase in successive generations in certain families; more repeats = worse phenotypeMay be in promoter, UTR, intron, or exonEx. Myotonic dystrophy: myotonia (takes longer to relax muscles), apathetic facial expression, cardioyopathy, MR, hypotonia in infants (can’t resist gravity)DMPK gene recomes much larger with CGT repeatsEx. ALS (amyotrophic lateral sclerosis): progressive m. weakkness, degeneration of motor neurons, dementia, frontal temporal lobe dementiaMay also be caused by successive shortening of telomeres (Ex. dyskeratosis congenital)Mosaicism: Presence of at least two cell lines in individual w/ different genotypesIn placenta: helps fetuses survive trisomies 18 and 21Somatic: causes patchy skin (ichthyosis), MR, cancer; proteus syndrome (elephant man; caused by missense mut. In AKT of mTOR pathway)Germline: Ex. Osteogenesis imperfecta and achondroplasia (dwarfism)Mitochondrial Inheritance: circular, small genome encoding 13 mt prot.s of ETC and RNAsMaternal inheritance; mutations may be homoplasmic (affect all mt genomes in cell) or heteroplasmic (cell has some wt and some mutant mt genomes)Disorders are hard to diagnose; pleiotropic (involves unrelated organs)Ex. Leber’s hereditary optic nueropathy (bilateral optic nerve death)Ex.MELAS (mt myopathy, encephalomyopathy, lactic acidosis, stroke)Epigenetics: mitotically heritable changes in gene expression not coded in DNAImprinting: transcriptional repression based on parent of originEx. Prader Willi Syndrome (won’t feed as infants, then execssive eating obesity, MR) caused by maternal imprinting of gene (SNRNP) and paternal deletion of chrom 15 bandEx. Angelman Syndrome (severe MR, gait problems) caused by paternal imprinting of a different gene (codes for E3 so proteins are not targeted for ubiquitnation/degradation) and maternal deletion of chrom 15. BandOnly in mammals (for placenta); loss of imprinting seen in cancers; may be tissue-specificDNA methylation of cytosinesHistone modification (involved in compacting DNA) may activate or silenceUniparental Disomy: both members of chrom. pair are from one parentDuplication of same chromosome from one parent: isodisomyBoth chrom.s from one parent: heterodisomy (b/c fertilized egg was aneuploid w/ n=24 chrom. then lost one chrom.)Lack of paternal allele of met gene implicated in Cystic FibrosisLeads to homozygosity for all genes on that chromosomes rare recessive disorders (if isodisomy)If subject to impriting as well, you may have disorders*Clopidogrel (Plavix) is a prodrug; requires conversion to active thiol metabolite by Cytochrome P450- Once active, plavix inhibits ADP-stimulated platelet aggregation by binding to the receptors- CYP2C19*2 associated with diminished response to Plavis and poorer outcomes (due to decreased activation of Plavix) VIII. Population Genetics: study of allele frequenciesHardy Weinberg eq’ns work when population is at equilibrium (only if random mating, no migration, and no new mutations)P + q = 1 and p2 +2pq + q2 = 1Used for autosomal recessive disorders; freq. of alleles for dominant = # affected individuals(Bb)/2*# total pop.; for X-linked, freq. = # affected males/# total males New mutations usually have little or no effect; small fraction are deleterious and tiny fraction are beneficialRecessive, deleterious mutations “hide” In the heterozygous formEx. Duchenne MD (X-linked): 1/3 exit gene pool in affected males, but 2/3 are still in females; new mutations account for replacing the 1/3Dominant deleterious mutations don’t last long in gene poolFounder effect: population with small # of founders some disorders will be more or less common relative to general populationInbreeding increases chance of expressing recessive disorders; at a population level, overtime, inbred populations will decrease recessive allele freq. compared to U.S.Increases variation (ex. dogs), but may decrease vigorCoefficient of inbreeding (F or α) = probability that both of an individual’s alleles came from single ancestral allele (Ex. ? for sibs and 1/16 for cousins)Outbred populations have wide genetic variation, even after many generations of mutation and selectionProblem with saving endangered species: variation low survival lowContinuous (quantitative) phenotypes also have variation b/c many genes (each w/ small effect) have additive effect on the trait and environmental variation smooths out the distributionEx. height, blood pressure, cholesterol levelsGenetic screening for carriers of disease alleles is difficult b/c some diseases have >50 genes affected, there’s wide variation in normal population, and new mutations constantly ariseSome mutations have selective advantge in hterozygote formEx. Sickle cell trait and thalassemia confer resistance to malariaFY gene encodes “Duffy antigen” which is used by Plasmodium vivax to enter RBCs and also confers resistance to malariaCCR5 receptor used by HIV to gain entry into macrophages; if person has deletion (homozygous), they are largely HIV resistantHeterozyg. CF may increase survival of diarrhea (less fluid production)Pharmacogenetics: how our genes affect the effectiveness of drugsProblems with drug Inactivation Isoniazid treats TB and is inactivated by acetylation; people with high activity of liver’s acetylating enz. have rapid excretion compared to othersMuscle relaxants (in anesthesia) are normally inactivated by pseudocholine esterase, but people who lack the enzyme have prolonged paralysisNormal doses of antidepressants become toxic in people w/ defect in liver cyt P450 enzyme that introduces hydroxyl groups into the drug for excretionAdverse drug reactionsPrimaquine (anti-malarial drug) stresses RBCs to decrease glutathione concentrations in G6PD deficient cells hemolytic anemia (RBCs destroyed)G6PD deficient cells themselves confer resistance ot malariaInhalation anesthetics may cause muscle rigidity and rapid increase in body temp. (maligant hypothermia) death; caused by mut. In ryanodine receptorDrug receptors are differentBeta-blockers given to patients w/ heart failure to block adrenaline’s effect on heart (via beta-adrenergic receptors); however, variation of beta-receptors lead to different responsesDividing population into more homogeneous groups increases chances of finding positive effect of drug with minimal side effectsABO Blood groups due to alleles coding for glycosyl transferases that modify antigensAntibodies formed in response to antigen exposure from foodBinding of IgGs to RBCs red cell destruction via complement systemType O are universal donors (b/c no antigens); Type AB are universal acceptors (b/c no antibodies); incompatible transfusions are rare but fatalHemolytic disease of the newborn occurs if 1) first baby is Rh+ (from father) and mom is Rh- during birth, placenta breaks and mom makes IgG antibodies; 2) baby and mom are ABO compatible (otherwise, the IgM antibodies for AB would have lysed baby’s RBCs at birth); 3) second baby is Rh+ mother’s IgG antibodies destroy baby’s RBCs b/c IgGs can cross placenta (but IgMs- AB antibodies cannot)Can also be caused if mother given transfusion of Rh+ bloodPrevented by giving mom injection of Rh+ antibodies before first birth so her immune system doesn’t create themDNA fingerprinting uses microsatellites (tandem repeats) to ID individualsIX. Genetics of CancerA tumor is simply a swelling; they can be non-neoplastic (cysts/hernias) or neoplastic:Benign: polyps, adenomas, and neviCircumscribed; can remove through surgeryMalignant (Cancer): carcinomas (epithelium), sarcomas (meschyme), and leukemiasInvades blood/lymph; not a smooth borderWaves of clonal expanstion: Mutation in one cell causes cell to grow and become the predominant cell in that area 2nd mutation occurs and now that cell becomes dominant cell etc. (usually 5-10 waves)Typical tumor have 20-80 mutations (may be up to 190 in melanomas and lung cancers); childhood tumors and leukemias are around 10 mut.sOnly a subset of these mutations (usually 5-10) are “drivers”; other are passengersTwo-Hit Hypothesis: Shown in Wilms’ tumor (pediatric kidney) b/c familial form has 10,000 x incidence, multiple tumors, occurs earlier, and only a small portion of cells become cancers (not affecting all cells in body)First hit: mutation on one chrom. removes tumor suppressor; second hit: loss of heterozygosity (removal of normal chrom.) OR second mutation to delete normal copy of tumor supressorFamilial form already has first hit; need environ. mut. for 2nd hitFused normal cell and tumor cell heterokaryon injected into mice Adding Chrom. 11 no tumor; therefore, tumor suppressor gene must be found on Chrom. 11Ex. Retinoblastoma (Chrom. 13, not 11)Isochormosome 17q: one chrom. 17 has two q’s instead of one q and one pCell birth:cell death ratio = S (exactly 1 in normal cells; <1 = atrophy and >1 = tumor)Avg. driver gene has S = 1.004Three types of Driver genesTumor supppressor genes inhibit growth (like a car brake)p53 tumor suppressor had wide mutation spectra (mutated via sunlight, aflatoxin, smoke): activated p21 (inhibits transition from G1 S) and activates PUMA (signal for apoptosis)With p53 mut., too much cell birth and not enough cell deathOncogenes normally stimulate gorwth (like an accelerator)DNA Tumor viruses (ex. papilloma, papovaviruses, adenoviruses, herpes)Like getting 4 hits, b/c you activate 2 oncogenes and inactivate 2 tumor supressor genesEndogenous genes that become oncogenes when mutatedEx. Philadelphia chromosome in CML (translocation b/w 9 and 22)bcl-abl tyrosine kinase overactive; gleevac interferes w/ TK by binding to ATP site so TK can’t P-lateRepair genes (stability/caretaker genes) normally limit mutationsmismatch repair, nucleotide excision repair, and base-excision repair genesX. Epigenetics: modifications of DNA or associated factors (other than changing the primary DNA sequence) that are maintained during cell divisionA. DNA Methylation: inversely related to gene expression; a. Essential for complex organismsb. CpG islands are consistent unmethylated region around promoters, and shores have differences in methylationHuman VMRs (Variable methylated regions) are stable over time; can distinguish obese individuals (up to 20% of cause of obesity)B. Chromatin modificationa. Position effect variegation (PEV): heterochromatin represes expressoin of euchromatin genes; Ex. white gene (makes fly eys red) is silenced when it’s closer to heterochromatin (on centromere)Histone tails are covalently modified after Xlation via methylation, acetylation, P-lation, ubiquitination may activate or repress gene expressionc. Polycomb and tritorhax prot.s stabilize Xcription repression or activation, respectivelyd. large organized heterochromatin K9-modifications (LOCKs)e. Detected by ChIP (chromatin imunoprecipitation): DNA cross-linked and sonicated, then precipitated C. Genomic imprinting: silencing a specific parental allele or chrom. in gamete or zygote differential expression (phenotype reversible when transmitted through other parent)Proven by gynogenetic offspring (2 female nuclei; important in intraembryonic develop.) and androgenetic offspring (2 male nuclei; important in extraembryonic growth)Ex: IGF2 promotes growth and is active if DNA seq. is methylated b/c enhancer can reach the promoter; H19 inhibits growth and in unmethylated DNA strands, CTCF binds to strand, blocking enhance from activating IGF2 and activating H19D. Disrupts phenotypic plasticitya. Ex. Rett syndrome: mut. in protein that recognize methylation, so gene that’s supposed to be silenced remains activeCancer caused by hypomethylation of oncogenes at shores and hypermethylation of tumor suppressor genes cancerous colon cells look like liver cells in terms of methylation“global hypomethylation” observed in all cancers activates genes involved in metastasis; similar to LOCKsEpigenetic epidemiology: epigenome can modulate genetic effect of disease; depends on environment, age, and genome itselfXI. Common TraitsWhen blood flow is progressively compromised, angiogensis happens; but, when blood flow acutely compromised, localized cell death (MI or stroke)Level of circulating lipids and cholesterol may determine rate of atherosclerosisAutosomal dominant inheritance of one large subset of hypercholestemiaEnvironmental factors (smoking and diet) play a big role tooStudy took survivors of MI; used mostly <60 years old to minimize environ.Study found correlation b/w age and cholesterol levels (more so for females)Found that family members of patients w/ MI had above avg. lipid contentPCSK9 gene decreases LDL receptor levels high serum LDL; w/ mutant allele, LDL receptor levels are increased low serum LDL (less CAD)Obesity remnant of evolution to help humans survive in near starvation (30% genetic)microcosm of GI bacteria differ b/w lean and obese individuals (cause or effect?)Hypertension similar because we evolved when salt retention was advantageousEpilepsy: mutations found in >70 genes (Na/K/Cl channels, GABA receptors)Diagnoses lumped together in this category, but really are a collection of various genetic disorders ................
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