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Session 91: Cell Cycle Basics
• Normal cell replication
o Replication is mitogen- (growth factor) dependent (only divides when told to)
o Replication is anchorage-dependent (must be anchored to extracellular matrcix)
o Replication is contact-inhibited – cells normally stop growing when available space is filled
o Cells are mortal – normally have a limited number of divisions before they die (telomeres shorten)
o Cell division
♣ Complex network known an cell-cycle control system or cell cycle clock governs progression through cell cycle
• The cell cycle
o Two most basic functions
♣ DNA replication (accuracy)
♣ Chromosomal segregation (each daughter receives copy of entire genome)
o How do cells duplicate their contents?
♣ Mitochondria are very plentiful – doubling number with each cycle is sufficient to ensure nearly perfect segregation
♣ Other organelles (ER, Golgi) break into small fragments which increases their chances of equal distribution – grow in size in daughter cells
o Cell-cycle times
♣ Vary between species
♣ Examples: intestinal epithelial cells – 12 hours; fibroblasts – 20 hours; human liver cells – 1 year
o Stages
♣ M phase: mitosis, about 1 hour
♣ Interphase: about 23 hours (S-phase, chromosome duplication about 11 hours)
• Regulation
o Checkpoints:
♣ G1: Restriction Point – start checkpoint in yeast, are there growth factor signals present? Has the cell grown sufficiently?
♣ S Checkpoint – DNA damage checkpoint, DNA replication halted if genome is damaged
♣ G2/M Checkpoint – entrance into M blocked if DNA replication is not completed
♣ Spindle Assembly Checkpoint (metaphase-to-anaphase transition checkpoint) – anaphase blocked if chromatids are not properly assembled on mitotic spindle
o Cell cycle is controlled by protein kinase complexes
♣ Cyclin-dependent kinases (Ckds) – ser/thr kinases, inactive unless bound to cyclins, phosphorylates proteins involved in cell cycle when active
♣ Cyclins – have no enzymatic activity themselves, are a regulatory (activating) subunit of Ckds that direct to target proteins
• G1-cyclins (D) – induced by mitogens, regulates the activities of G1-Cdks (needed to go through restriction point)
• G1/S-cyclins (E) – help trigger progession through restriction point
• S-cyclins (A) – bind Cdks right after restriction point, stimulate chromosome duplication and control many early mitotic events
•
M-cyclins (B) – activates Cdks that stimulate entry into mitosis
♣
Regulation of Cdk Activity
• Controlled degradation of cyclin subunits, some Cdks degraded during cell cycle (each cyclin only present during specific time during cell cycle)
• Phosphorylation and dephosphorylation regulates activity
• Cdk-inhibiting proteins (CKIs, CIPs, INKs) interfere with kinase activity
• Transcription of the cyclins and CKIs
o Controlled degradation of cyclins during the cell cycle
♣ Cyclins “rule the cycle” – undergo a cycle of synthesis and degradation
♣ Cycle ensures that
• Each checkpoint is ‘checked’ at each cycle
• Only one round of cell division occurs unless mitogens still present
♣ During G1 and S phases, the SCF E3 ubiquitin ligase complex polyubiquitylate and destroys G1/S cyclins (D, E, A)
♣
In M phase, anaphase-promoting E3 ligase complex (APC/C) polyubiquitylates and destroys M-cyclins (B)
o Phosphorylation/dephosphorylation of the Cdks (ex M-Cdk)
♣ Mostly used to regulate M-Cdk activity
♣ MPF: M-Cdk in vertebrates
♣ Wee1: tyrosine kinase
♣ CAK: Cdk-activating kinase
♣ Cdc25: protein phosphotase
o CKIs Inhibit Cyclin-Cdk Activity
♣ CKIs wrap themselves around cyclin-Cdk complex to inactivate it
♣ Ex: p16Ink, p15Ink, p18Ink, p19Ink, p21Cip, p27Kip
o
Transcription of key genes regulates the cell cycle
♣ Transcription of cyclins, CDKs, etc
Session 92: Regulating the Cell Cycle
• Cyclin-Cdks required by ALL cells
o G1/S Cyclin-Cdks (cyclin E-Cdk): control entry into S phase (progression through restriction point)
♣ Phosphorylates transcription factors controlling genes whose proteins are needed for DNA replication
o S-Phase cyclin-Cdks (cyclin A-Cdk): controls DNA synthesis
♣ Phosphorylates protein components of the prereplication complexes at origins or replication
o Mitotic Cyclin-Cdks (cyclin B-Cdk): control mitosis
♣ Phosphorylates hundreds of proteins
• Fourth class required by MOST cells
o G1 cyclins (cyclin D-Cdk): control the activities of G1/S cyclins
♣ Induced by mitogens
♣ Note: mitogens not required by embryonic stem (ES) cells
• ES cells respond to an intrinsic timer or oscillator instead of mitogens
• Don’t really have a restriction point
• Only WT cells that are tumorigenic
• Cell Cycle ‘Clock’
o Integrates signals from outside and within cell to control cell cycle initiation and progression
♣ Signals include tyrosine kinase, GPCRs, TGFB, nuclear receptors, nutrient status
• G1 Checkpoint:
o M-cyclin (cyclin B) degradation and Cdk Inactivity ends M-Phase and begins G1
o Key point where cells decide whether or not to divide
o Based on whether mitogenic signals present, DNA damage, cell has grown sufficiently
o Cells enter G0 between divisions
♣ May be terminal (neurons, muscle cells)
♣ Can be quiescent state but one where cell cycle machinery is intact (liver)
♣ Can by transient, cells enter and leave rapidly (fibroblasts, intestinal cells)
♣ Imposed on all cells, at least temporarily, by degradation of the M-phase cyclin after mitosis
o Mitogens stimulate synthesis of the D-cyclins (MAPK, JAK/STAT, Wnt pathways, etc), induction of Myc, AP-1 (Fos and Jun), B-catenin, STATs, SP1…
♣
Inactivation of Rb and activation of E2F
♣ Induction of SCF and degradation of CKIs
♣ Synthesis of G1/S cyclins and other proteins needed for DNA synthesis
♣
♣ When TGFB is acting as a tumor suppressor, it opposes cell proliferation by preempting the mitogenic pathway
• Increases expression of CKIs
• Blocks phosphorylation of Rb
• Prevents expression of Myc
o Embryonic Stem ells are not subject to the G1 checkpoint
♣ Rb is usually hyperphosphorylated all the time and thus inactive
♣ Mitogens and MEK signaling not needed for progressiong G1
♣ No DNA damage checkpoint in G1
♣ Cyclin E expression is constant, not cyclical
♣ Net result: ESC pass through G1 rapidly, allowing rapid cell proliferation
• DNA damage checkpoints (G1/S-Cdk, S-Cdk, M-Cdk phases)
o DNA damage (esp single- double-stranded breaks) is detected by Kinases
♣
Activate the ATM and ATR kinases
♣ ATM and ATR activate the Chk1 and Chk2 kinases
• These phosphorylate Cdc25 phosphatase leads to degradation
• Cdc25 normally removes inactivating phosphoryl from Cdks, without this signal Cdks cannot be activated cell cycle is blocked
♣ Both ATM and Chk kinases increase p53 levels by phosphorylating p53, which kicks off inhibitor Mdm2 (remember???)
• Binds to many target genes
• Signals DNA repair
• If this fails apoptosis
• Inhibits cell cycle (CKI activation)
• Unreplicated DNA Checkpoint (M-Cdk phase)
o Also activates Chk1, inactivates Cdc25, etc
• Spindle-Assembly Checkpoint (APC/C)
o
APC/C activated degrades Securin (from Securin-separase complex) active separase cleaves cohesins that hold chromatids together
o Prolonged activation of the checkpoint cell death
o Many anti-cancer drugs target this step
Session 93: the Cell Cycle and Cancer
• One check against mitogen overstimulation: increased p53
o Excessive Myc production activates Arf (14-3-3) complexes with Mdm2 (inhibits) increased p53
• Cells can overcome their control systems
o Cancer from deregulated cell proliferation
♣ Mutations that short-circuit the need for mitogens – activating proto-oncogenes (RTKs, Cyclin D, Ras, PI3K, etc); or inactivating tumor suppressors (PTEN, p53, TGFB)
♣ Mutations that target the G1 checkpoints – inactivation tumor suppressors (Rb, CKIs, p53); mutations that increase Myc or AP-1
o Cancer from deregulated cell survival
♣ Mutations that suppress apoptosis – activating mutations in PI3K cascade, inactivating mutations in PTEN, p53
• Mutations of critical genes
o Gain of function of proto-oncogene creates ocogene – dominant only in somatic cells
o Loss of function of tumor suppressor genes – (typically, not p53) recessive in somatic and germ cells (can be heritable), can have strong tissue preference
• Common conversion of proto-oncoproteins oncoproteins
o Missence mutation in transmembrane regions of Her2/neu receptor leads to dimerization even in absence of ligand
o Deletion of external domain of EGF receptor dimerization without ligand
o Chromosome translocations can create fusion proteins with oncogenic proteins
♣ Philadelphia chromosome created by translocation of tips of 9 and 22
♣ Creates BCR-ABL fusion protein, which is a constitutively active tyrosine kinase
♣ Phosphorylates many signaling molecules, such as JAKs
♣ Leads to chronic myelogenous leukemia (CML) if occurs in bone marrow
♣ Imatinib (Gleevac) targets Abl kinase first cancer drug targeting to a signaling protein unique to cancer cells
• Mechanisms for inactivating a tumor suppressor gene
o Rb gene example (major tumor suppressor for cell cycle progression)
o All except point mutations lead to LOH
o Tumor suppressor genes can be inactivated by both epigenetic and genetic mechanism
♣
Epigenetic changes are much more common could inactivate only good copy of gene
•
Why isn’t cancer more frequent?
o 1016 cell divisions occur in humans in a lifetime
o Spontaneous mutations in a carcinogen-free environment occur about 10-6bp/gene/cell div
o In a lifespan, each gene is likely to have been mutation 1010 times
o Multiple genetic events are needed
o Cancers are though to arise from a single cell with more than one mutation
• Evidence supporting Multi-Hit Model of cancer induction
o All of the cells in a tumor should have at least some genetic alterations in common
♣ Supported by microarray analysis; in female tumors all cells have same X-inactivation
o Cancer incidence increases with age more chance for multiple mutations to occur
o In mouse models, even with overexpression of potent oncoproteins, cancer initiation is extremely slow unless more than one is introduced
o Successive mutations have been traced in colorectal cancers
• Mutations in p53 are particularly devastating – ‘guardian of the genome’
o Reponds to many different signals (lack of nucleotides, UV damage, hypoxia, etc)
o Promotes cell cycle arrest when DNA is damaged
o Triggers DNA repair mechanisms
o Initiates apoptosis when damage is too severe
o Blocks angiogenesis, excessive mitogen signaling
o p53 functions as a tetramer, mutations in only one allele of TP53 can cause cancer
♣ Mutations that inactivate p53 usually occur in DBD typically recessive, usually require both alleles to be mutated, but in some cases can bind to WT p53 in dominant fashion
♣ Other mutations are in the oligomerization domain dominant negative mutations, only one allele needs to be affected
♣ Ex: Li-Fraumeni syndrome – individuals with this disease develop tumors early in life (AD)
♣ 50% of cancers have mutations in p53, other 50% have mutations in p53 regulators (Chk1)
• Characteristics of Cancer Cells
o Abnormally high mitotic rate - used to estimate malignant potential
o Signs of de-differentiation and assume features of immature stem cells
o Disordered growth – not subject to contact inhibition and grown in a sprawling mess
♣ Anaplasia – de-differentiation and disordered growth, degree of anaplasia is predictive of malignant behavior, patient’s survival
o Can metastasize – ability to migrate, cross basement membrane, grow in a strange environment
o Are genetically unstable – keep mutating because they cannot prevent or repair DNA damage
o Are immortal
o Become self-sufficient for growth and proliferation
o Induce help from local stromal cells
o Induce angiogenesis
Session 95: Cancer Genetics
• Cancer Basics
o Common – 1/3 of population will have cancer in lifetime, 20% of deaths in developed nations
o Early diagnosis improves outcome
o Cancer causes (sporadic most common)
♣ Sporadic – frequently only one person in family with cancer, no germline mutation
• Typically occur later in life
♣ Familial – number of primary/secondary relatives with cancer, no evidence of a cancer syndrome or germline mutation
♣ Hereditary – heritable germline mutation, inherited in autosomal dominant manner
o Multistep process, requires accumulated sequential mutations
• Oncogene
o Mutations in genes that normally function to promote cell survival or limit cell death
o Examples: telomerase, Bcl2, Myc
o Typically cause cancer by a gain of function mutation
• Tumor Supressor Genes
o Gatekeepers – control cell growth by regulating cell cycle checkpoints or promoting apoptosis
o Caretakers – guardians of the cell’s genome, correct normal day to day errors that occur in genome
o Typically cause cancer by loss of function mutations
• Hereditary Cancers
o Autosomal Dominant
o Penetrance is not 100% but quite high
o If the mutation is in a tumor suppressor gene, requires a second hit for the cancer to start
o Frequently cell type (cancer type) specific but may have patterns of tumors that would be indicative of the cancer syndrome
o Tend to be bilateral in paired organs (breast cancer, retinoblastoma)
o Tend to be early onset
o Hereditary 54 described hereditary cancer syndromes
• Retinoblastoma
o Caused by germline mutations in the Rb gene, requires second Rb mutation to develop tumor
♣ Rb encodes a gatekeeper
♣ Inherited in dominant manner, but in the cell it is recessive (as second mutation must happen)
o Clinical presentation
♣ Very early onset: sometimes present at birth
♣ Tumors can be bilateral and at multiple sites in retina
♣ Later in life, individuals are at risk for osteosarcoma
• Colon Cancer Syndromes
o Familial Adenomatous Polyposis (FAP)
♣ Autosomal dominant mutations in APC gene controls action of beta-catenin in Wnt pathway
♣ Diagnostic charactertics
• >100 colorectal adenomatous polyps (typically in 1000s)
• <100 and a relative with FAP
• Attenuated (milder) forms may have fewer polyps
• Onset of polyps early in life; median age is 16 years (range 7-36 years)
♣ Polyps are first benign progress to cancer
♣ >95% with APC will develop cancer
♣ 12% will develop duodenal carcinoma
♣ Screening (sigmoidoscopy), many individuals eventually have colon removed
o Lynch Sydrome (Hereditary Non-polyposis Colon Cancer, HNPCC)
♣ Early appearance of colon cancer (average is 43, sporadic is typically early 60s)
♣ Family history of colon/endometrial cancer
♣ Cancers grow quickly and are aggressive
♣ Mutations in four genes: MLH1, MSH2, PMS2, MSH6 (possibly MSH3, PMS1)
♣ Genes involved in mismatch repair
• Hallmark ‘microsatellite instability’ – DNA slippage events in short repeat units are not repaired, show slightly different microsatellites sizes when run on gel
♣ Cancers in lynch syndrome
• Colon cancer: tends to be ‘right-sided’, risk of 70% by age 70
• Endometrial cancer: 30-60% by age 40
• Other tumors with a high percentage: gastric, biliary, ovarian, urinary tract, etc…
• Requires screening with consideration of removal of colon, uterus, ovaries
• Li-Fraumeni Syndrome
o Caused by mutations in p53 (TP53)
♣ TP53 causes cell cycle arrest that is required to permit repair of damaged DNA
o Clinical presentation
♣ Early onset cancer syndrome with multiple primary cancers throughout lifetime
♣ 90% incidence of cancer by age 70
o Cancers
♣ Breast cancer 25%
♣ Rhabdomyosarcoma 20%
♣ Brain cancer 13%
♣ Adrenocortical carcinoma 10%
♣ Osteogenic, chondrosarcoma 15%
• Hereditary Breast Cancer Syndromes
o BRCA1 and BRCA2 mutations found in 2/3 of families with 3+ individuals with breast/ovarian cancer, found in 10% of women regardless of family history
o High frequency of BRCA1/2 founder mutations in Ashkenazi Jewish women
o Lifetime risk of breast cancer (men are also at risk for breast cancer)
♣ BRCA1 by age 70
• 65% will have breast cancer, 39% will have ovarian cancer
♣ BRCA2 by age 70
• 49% will have breast cancer, 18% will have ovarian cancer
o Role of BRCA1/2
♣ BRCA1 – multifunction E3 ubiquitin ligase involved in DNA damage, signaling, DNA repair by homologous recombination
♣ BRCA2 – repair of double stranded breaks through homologous recombination
o Cancer screening
♣ Monthly self examinations starting at age 18
♣ Clinical examination twice yearly starting at age 25
♣ MRI and mammogram yearly starting at age 25
♣ Twice yearly transvaginal ultrasound/CA125 blood test (ovarian cancer marker)
♣ Consideration of salpingo-oophorectomy after age 35-40 or childbearing
♣ Consideration of prophylactic bilateral mastectomy
Session 97: Ethics and Genetic Counseling
• Ethics
o Establishment of a set of guidelines for morally acceptable conduct within a theoretical framework
o Principles
♣ Beneficence – benefit
♣ Nonmaleficence – no harm, for example doing research/clinical trials
♣ Autonomy – rule of self, patient makes independent decisions
♣ Justice – equal care
• Ethical/professional issues in genetics
o Informed consent – to make a decision, patient needs to understand treatment, other options, privacy issues, etc…
♣ Examples: predictive testing for adult onset diseases (ALS, BRCA), testing minors, research
o Confidentiality – situations in which a professional must decide whether or not to share privileged information with family members, insurance companies, fellow professionals, third parties
♣ Example: duty to warm family members of risk, must counsel patient about this
o Withholding information – situations in which a professional must decide whether or not to share information with patient
♣ Examples: unanticipated information (non-paternity, whole genome sequencing), withholding is in patient’s best interest?
o Uncertainty – situations where there is ambiguity about how genetic information
♣ Examples: test results from cancer susceptibility test (lack of data), variants of uncertain significance
o Value Conflicts - situations where there is a difference in a personal/professional values
♣ Examples: sex selection, patients selecting for such traits as deafness of dwarfism
o Directiveness – to what extent should a professional influence a patient’s decision making
o Discrimination – situations in which a professional or patient is concerned about unfair treatment by an insurance company or employer based on results of a genetic test
♣ MN statute and GINA protect in some circumstances
o Diversity – situations in which ethnicity, religion, socioeconomic status, language etc present an obstacle in the genetic counseling process
o Who is the patient? – situations in which parents disagree about what to do with respect to child or families disagree about procedures/testing
o Documentation – illegal not to record, even if patients doesn’t want it to be
Session 99: Gametogenesis and Fertilization
• Sexual Reproduction
o Advantages: Genetic variation and elimination of deleterious genes (through sexual selection)
o Timing, onset, duration of meiosis are sexually dimorphic
• Retinoic Acid (vitamin A derivative) regulates the timing of entry into meiosis
o RA binds to RAR, a nuclear receptor
o Females: in embryonic ovary, high levels or RA induce proliferating germ-line cells to enter meiosis at about 11-12 weeks of gestation
o Males: in embryonic testis, sertoli cells produce an enzyme (Cyp26b1) that metabolizes RA, only after puberty do enzyme levels decrease, allowing RA levels to rise and meiosis to resume
• Gametes specialization
o Sperm: very small, highly motile, no organelles except for mitochondria, highly competitive
o Ovum: very large, nonmotile, contains many materials/organelles to support embryo growth
•
Spermatogenesis
o Sperm develop as a syncytium – connected together to synchronize development and so X containing sperm get same proteins as Y containing sperm
o Acrosomal vesicle contains hydrolyic enzymes to penetrate ovum’s outer coat
o DNA is tightly condensed by protamines and sperm-specific histones
o Sertoli cells (nurse cells)
♣ Secrete anti-Mullerian hormone (AMH)
♣ Convert testosterone to estradiol
♣ Form blood-testes barrier
♣ Secrete inhibin and activins after puberty to regulate FSH secretion
o Leydig Cells (interstitial cells)
♣ Produce testosterone in response to LH
o Sperm are not capable of fertilization until they undergo capacitation in the female genital tract
♣ Occurs in fallopian tubes, takes 5-6 hours
♣ Involves extensive biochemical and functional changes in the sperm
• Cholesterol efflux, membrane becomes hyperpolarized, increased cytosolic pH, unmasking of cell surface receptors that bind the sperm to the ovum
♣ Stimulus for capacitation is unclear, in vitro fertilization requires albumin, Ca2+, HCO3-
• Albumin helps extract cholesterol, increasing the ability of plasma membrane to fuse with acrosome during acrosome reaction
• Ca2+, HCO3-Eenter the sperm and activate a soluble adenylyl cyclase enzyme to increase cAMP leads to tyrosine phosphorylation of many proteins
•
• Ovum maturation
o Oocyte: developing egg/ovum that cannot bind sperm of be fertilized
o Egg = mature ovum = ovum: capable of being fertilized, arrested in metaphase II
o The primary oocyte uses special strategies to grow:
♣ Most of the growth occurs when the cells are bivalent (4N), so there is twice as much DNA to use for RNA synthesis
♣ Some genes such as ribosomal RNA genes become amplified
♣ Follicle cells link to oocyte via gap junctions to provide precursors for protein synthesis
o Ruptured follicle corpus luteum secretes progesterone to maintain pregnancy
o
Gonadotropins FSH and LH regulate Ovarian and Testicular Development
♣ During reproductive years, LH and FSH stimulate ovulation
♣ During puberty, Kisspeptin is the key regulatory of the onset of puberty
• It binds to a GPCR in the pituitary (GPR54)
• This signals the pituitary to release LH and FSH
• FSH drives maturation of Sertoli, Granulosa cells
• LH stimulates Leydig, thecal cells
o Regulation of Meiosis in Mammilian Oocytes
♣ Meiotic arrest depends on high cAMP levels in oocyte
♣ The FSH/LH surge acts on granulosa cells to trigger the oocyte to resume meiosis
♣ CNP and NPR-B (natrieretic peptide receptor) contribute to maintaining meiotic arrest
•
NPR2 GTP to cGMP inhibits PDE3A stops degradation of 5’-AMP
• Fertilization
o Around 300 million sperm are deposited at the opening to the cervix
o Only about 200 reach the ovum
o Ovum surrounded by glycoprotein matrix called zona pellucida and layer of follicular cells called corona radiata
o Meeting of sperm and ovum
♣ Ovum secretes chemoattractant peptides
♣ Sperm binds to the zona pellucida
♣ Sperm passes through ZP, contains glycoproteins ZP1, ZP2, ZP3 (may be sperm receptor)
♣ ZP induces the acrosomal reaction, contents of the acrosome are exocytosed in a Ca2+ mediated reaction
o Blocking Polyspermy
♣ Fast reaction: electrical potential of the ovum membrane is changed within 1-3 seconds as Na2+ enters ovum lasts 1 min (not detected in mammals)
♣ Slow reaction: the cortical granules fuse with the ovum membrane, modify proteins in the ZP including ‘sperm receptor’ ZP3 cortical reaction
o Ovum activation after sperm binding begins development program (involves PLC pathway Ca2+ ^^)
♣ Exocytosis of cortical granules
♣ Resumption of meiosis and extrusion of 2nd polar body
♣ Release of inhibition on maternal mRNAs
♣ Stimulation of protein synthesis, DNA replication, etc
o Sperm contributes centrioles needed for first mitotic division
Session 100: Sex Determination and Differentiation
• Primordial Germ Cells (PGCs)
o PGCs are the cells that give rise to the gametes
o PGC specification begins at gastrulation, initiated by high levels of BMP family members (in mouse)
♣ Part of TGFB family, bind to Ser/Thr kinase receptors
o Specification: the first stage of commitment of a cell or tissue to its fate
• Mammalian Gonad
o Bipotential (indifferent) – can become male or female from weeks 4-7
o Sex chromosomal composition of the somatic cells surrounding the PGCs determines differentiation
o Male: wolffian duct, XY or XXXXXXXXXXXY
o Female: Mullerian duct, XX or X0
• Development of Male Phenotype
o Testis determination controlled by SRY (sex determining region of the Y chromosome) on short arm of Y chromosome
♣ Encodes 223 aa tranction factor, expressed in a number of tissues in fetal development
♣ Activates the male-specific transcription factor Sox9
♣ Somatic cells expressing Sry gene become sertoli cells
o Sertoli Cells direct development
♣ Stimulate PGCs to develop along pathway that produces sperm and inhibit from entering meiosis, which oocytes must do
♣ Secrete AMH (anti-Mullerian hormone) causes regression of Mullerian duct
♣ Stimulate development of other somatic cells to become Leydig cells
♣ Leydig cells secrete testosterone, induces male reproduction structures and secondary sexual characterstics and masculinizes the brain
o
Inititation of sex determination
♣ In the default female pathway, paracrine signals and transcription factors activate Wnt4 and R-spondin1
• Wnt4 increases beta-catenin, which inhibits Sox9 male pathway cannot proceed
♣ If Sry is present, it acts with Sf1 to induce Sox9
• Sox9 induces Fgf9, stimulates testis development and feeds back to increase Sox9
o Fgf9 – growth factor, binds to tyrosine kinase receptor
• Sox9 inhibits beta-catenin’s activation of ovary
o Mechanism of action of Sry
♣ Contains two nuclear locatization domains, must be bound by CaM and Impbeta to move into nucleus
♣ Sry and Sf1 bind directly to enhancer region (TESCO) of Sox9 gene induce expression of Sox9
♣ Sox9 also binds directly to TESCO site with Sf1 to amplify Sox9 expression
o Sox9: the autosomal testis-determining gene
♣ Transcription factor that induces testis formation in all vertebrates
♣ Functions
• Upregulates its own promoter, prolonging its own expression
• Blocks ability of beta-catenin to induce ovary formation
• Regulates numerous genes requires for testis formation – AMH, FGF9
♣ NOTE: although Sry is the trigger for male sex determination, Sox9 orchestrates and stabilizes Sertoli cell differentation to lock in the testis-determining program
o FGF9 is also essential – induces proliferation of Sertoli cell precursors and stimulates their differentiation, without sertoli cells, the male structures do not form
o Sf1 (steroidogenic Factor 1) – link between Sry and male development pathways
♣
Nuclear orphan receptor (ligand not known)
♣ Binds to DNA in monomeric form
♣ Activates AMH in sertoli cells and the enzymes that make testosterone in Leydig cells
♣ Individuals heterozygous for Sf1 have malformed gonads, retain Mullerian duct
• Development of Female Phenotype
o Initiation of sex determination (see figure above)
♣ Female pathway is default, Wnt4, Rspo1 (inhibits Wnt inhibitor)beta-catenin inhibits Sox9
• Beta-catenin prevents Sf1 from binding to TESCO enhancer
♣
Wnt4, R-spondin1 required for normal ovarian development
•
Sex determination vs. differentiation: development of female and male phenotypes in response to hormones secreted by the gonads
Session 101: Disorders of Sex Development
• Disorders of sex development
o Genotypic sex based on presence of the type of sex chromosomes and the phenotypic sex are disconcordant or external genitalia are sufficiently ambiguous to preclude sex assignment at birth
o Umbrella term that supplants terms like intersex, hermaphrodite, etc…
o Many are characterized by ambiguous genitalia: hypospadias, chordee, absent testes, enlarged clitoris, masculinized labia majora, extrophic bladder
• Terms
o Wolffian structures: male structures – epididymis, vas deferens, seminal vesicles
o Mullerian structures: female structures – fallopian tubes, uterus, upper 2/3 vagina
• Sex Chromosome Disorders
o
Turner Syndrome
♣ 45, X (most common); 46, X, iso Xp (has two short arms); 46, x, iso Xq; X translocation to an autosome or Y chromosome, 45, X/46, XY mosaicism (ratio determines sex characteristics)
♣ 1/5000 live births
♣ Clinical characteristics:
• Short stature, infertility, congenital heart disease, streak gonads (no follicle cells in ovaries), kidney malformations, hand/food edema, neck webbing, wide carrying angle, low hair line, wide spaced nipples, late onset hearing loss
o Klinefelter Syndrome
♣ 1/500 males
♣ 47, XXY
♣ Clinical characteristics
• Tall stature with long limbs, small testes, low testosterone – leads to under-virilization and decreased bone density, azoospermia, learning disability or behavioral changes
o 46, XX/46, XY Chimerism
♣ Two cell lines that occurred from different zygotes
♣ Phenotype depends on percent and location of one cell type vs another
• 46, XY Disorders
o Partial and Mixed Gonadal Dysgenesis (PGD)
♣ Characteristics
• Ambiguous genitalia, mild to severe penile scrotal hypospadias, dysgenetic testes may become tumors later in life, reduced to absent sperm production, various degrees of both Mullerian and Wolffian structures
o Complete Gonadal Dystenesis (CGD)
♣ Individuals appear as normal woman and may not present until puberty when they fail to develop menses
♣ Completely underdeveloped gonads, need to be removed by adulthood to prevent tumors
♣ Normal Mullerian structures
♣ Woman are infertile, but may carry a donated pregnancy
o Genetic basis
♣ Deletions of small regions on 9p, 2q, 10q
♣ Duplications of 1p (Wnt4) or Xp (Dax1 aka NROB1)
♣ Single gene mutations
• Sry – 1% of 46, XY PGD; 15% of 46, XY CGD
• Sf1 – 13% of 46, XY DSD
• DHH – 20% DSD, 50% CGD
• WT1
• 46, XX Disorders
o Testicular Disorder of Sex Development
♣ 46, XX Karyotype
♣ clinical findings
• male appearance (80%) or ambigiuous male (20%), two testicles, no Mullerian structures, low testosterone, gynecomastia
♣ Genetic basis
• Translocation of Sry from Y to X chromosome found in majority
• De novo event
o Ovotesticular Disorder
♣ Formerly known as True Hermaphroditism
♣ Causes are variable: chimerism – 46, XX/46, XY; mosaicism, etc
♣ Characteristics
• Mullerian and Wolffian structures may both be present
• Hormonal basis of DSD
o Abnormalities of sex differentiation
♣ In production or response to hormones that are produced by the testes, adrenal glands, or other sources that change the external appearance of genitalia
o Congenital adrenal hyperplasia (CAH)
♣ Mutation in genes that encode enzymes needed for the biosynthesis of cortisol
♣ Most common is 21-hydroxylase deficiency
♣ Have increased 17-hydroxyprogesterone (used to screen)
♣ Characterstics: virilized female genitalia – ambiguous genitalia appears male
♣ Infants can have salt wasting – leads to death in first days of life if not treated
♣ Common – 1/10000 in Europe, 1/300 in Yupik Eskimos
o 5 alpha-reductase deficiency
♣ 46, XY
♣ Ambiguous genitalia, testes present in abdomen or inguinal canal or labia, virilization at puberty due to effect of testosterone
♣ Most individuals are raised as girls but adopt male identity after puberty
♣ Testosterone DHT by 5-alpha reductase, induces transcription of proteins needed for male differentiation
o Complete Androgen Resistance
♣ 46, XY but unambiguous female
♣ Mutation in gene encoding the Androgen Receptor in Xq11-12
♣ Testosterone is produced but cells cannot respond to its signal
♣ 2-5/100000 individuals (fairly rare)
♣ Individuals have normal breast development at puberty, testes present in abdomen or inguinal canal or labia
♣ Most individuals raised as women
• Sex assignment
o 2/10000 babies are born with ambiguous genitalia
o Both a medical and a social crisis
o Do not risk assignment until you have all the data you need to help the child
o Testing needed:
♣ Karyotype, steroid measurements, electrolytes to rule out salt wasting, blood pressure, ultrasound or MRI of pelvis
o Final assignment may take days, and genotypic sex may not be the same as phenotypic sex
• Surgical Management
o Controversial intervention
o Arguments against early surgery
♣ Young girls have no use for ‘functional’ vagina until menses, intercourse
♣ We could reduce total number of operations needed to achieve vaginal length, while reducing risk of stenosis and give patients greater control over their lives
♣ No evidence that early surgery improves gender or psychosocial development
o Arguments for early surgery
♣ There is more mobility of infant tissue
♣ There is a shorter pelvis length that allows easier mobilization of urogenital sinus
♣ Many still argue that psychosocial rearing/bonding with parents is easier (no evidence)
o Consensus
♣ Surgery recommended for patients with high confluence by 2-6 months of age
♣ Never remove clitoris, preserve neurovascular bundles
♣ Females with CAH have low risk for gender identity problems
o Evaluation before surgery
♣ Defining vaginal confluence with UG sinus is most important step
♣ Genitography is recommended for pre-operative planning
o Procedure
♣ Goals are to recreate normal appearance and function external genitalia
♣ Preserving bladder function
Session 102: Genetic Association Studies
• High sensitivity = genotype catches all cases, Low Specificity = many unaffected have same genotype
• Low sensitivity = miss the correct genotype for cases, High specificity = avoid misdiagnosing unaffected
• Clinical utility
o Is it sensitive and specific?
o Does it provide predictions of disease occurrence and outcomes?
o Does it replace or contribute to other clinical diagnostic tests?
o Is it cost effective?
• What tools do we have?
o Genome variations associated with disease (DNA)
♣ Genome Wide Association Studies – GWAS
• Manhattan plot shows SNP association with disease vs control
• Pros: high throughput screening with no needed hypothesis, use of haplotype blocks and LD can simplify search, can show significant association even if low penetrance
• Cons: multiple testing error = 1/20 association may occur by chance, need to overcome false positives by large case-control numbers, may identify a haplotype block not a gene or specific cause, OR may be low association, may have significant association and NO UTILITY
♣ Make use of HapMap information
o Gene expression profiling (mRNA)
o Whole genome sequencing
Session 103: Cancer Cytogenetics
• Constitutional Abnormalities – germline; in most cases, arise during meiosis
• Acquired Abnormalities – acquired in association with the development of a malignant process (sometimes in utero, usually after birth)
• Most cancer cells have associated chromosomal abnormalities
o Abnormalities are acquired, clonal (two or more cells have the same abnormality), limited to the tissues involved in the malignancy
o Identification of abnormality is important for diagnosis, (gene-product targeted) therapy, prognosis
• t(9;22) translocation
o Example karyotype: t(9;22)(q34;q11.2)
o Diagnosis: Chronic myelogenous (myeloid) leukemia (CML)
o Derivative chromosome 22 is called Philadelphia chromosome Ph+
o Translocation results in fusion of the breakpoint cluster region (BCR) in 22q11.2 to the Abelson murine leukemia virus oncogene (ABL1) in 9q34
♣ BCR-ABL1 fusion on 22 is Philadelphia chromosome
♣ ABL1-BCR on 9 is not involved in leukemia
♣ Can be detected by FISH
♣ Translocation results in the formation of a novel (chimeric) BCR-ABL gene that encodes a protein with altered tyrosine kinase activity
o Cytogenetics used in CML
♣ Confrism diagnosis, determine stage, monitor response to therapy
o Natural history of CML
♣ Chronic phase: t(9;22) is typically the sole abnormality, symptoms very mild
♣ Accelerated phase: 75% of cases gain additional abnormalities (e.g. +8, i(17)(q10), +der(22)t(9;22)), secondary abnormalities are no consistent but some are commonly seen
♣ Blast crisis: additional abnormalities typically present as in accelerated phase, lethal if not treated
o Current therapies
♣ Hematopoetic stem cell transplant
♣ Gleevec - Therapy targeted at the specific abnormal tyrosine kinase generated by the t(9;22)
• Don’t know if individuals need to take therapy their whole lives
• t(15;17) translocation
o Example karyotype: t(15;17)(q24.1q21)
♣ PML (promyelocytic leukemia) gene on 15q24.1 fuses with RARA (retinoic acid receptor alpha) on 17q21
♣ RARA transcription factor regulating transcription of genes important in the maturation of white blood cells beyond the promyelocyte stage
♣ PML acts as a tumor supressor
o Diagnosis: Acute Promyelocytic Leukemia
♣ Arrest in development of promyelocytes, have fibrous-looking rods in cytoplasm
o Recognition of the PML-RARA fusion is critical for care
♣ APL associated with coagulopathy
♣ Targeted therapy: ATRA + Arsenic + chemo
♣ Recent study in blood: 98% sustain complete remission
♣ ATRA should be given within 24 hours of diagnosis – given to patients when PML is suspected
• t(4;11)(q21;q23) translocation
o Presentation: somewhat elevated WBCs, very high circulating blasts, anemia
o A specific recurring abnormality
♣ Accounts for 60% of cases of acute lymphoblastic leukemia of infancy
♣ Very poor prognosis – immediate planning for bone marrow transplant
o MLL gene is a human homolog of Drosophila trithorax gene that regulates HOX genes
o AF4 is thought to be involved in lymphocyte development
• High hyperdiploidy (>52 chromosomes)
o Associated with B-cell lineage acute lymphoblastic leukemia of childhood
o Very favorable prognosis
o Presence of trisomies for 4,10 now used to stratify to a ‘low risk’ leukemia therapy group
• Case 5: 22 yr old female, B-acute lymphoblastic leukemia
o Presentation: hypercellular marrow, very high blasts, B markers present, normal G-banding cytogenetics, normal FISH for the recurring abnormalities
o Array showed 21 abnormalities not shown through G-banding
o Found 7 important abnormalities
♣ Small deletion in 7p12.2: IKZF1 gene
• Associated with increased risk of relapse and adverse events
• 74% will relapse
♣ Small deletion in 9p13.2: PAX5 gene
• Recent studies show some patients with high-risk, active lymphoblastic leukemia have mutations affecting tyrosine kinase and cytokine signaling can be targeted
• HER-2/neu
o c-erb B2
o A proto-oncogene
o Encodes a tyrosine kinase receptor
o The ligands of HER-2/neu and related growth factor receptors are known as heregulins
o HER-2/neu amplification
♣ Pts with multiple copies of the gene in tumor tissue had a shorter time to relapse and a shorter overall survival
♣ Amplification occurs in 25-30% of human breast cancers, also in some ovarian malignancies
o Therapy
♣ Use of recombinant anti-HER-2 monoclonal Ab (Herceptin/trastuzumab) together with cisplatin clinical response in patients with HER-2 overexpressing metastatic breast cancer refractory to other chemotheraputic regiments
♣ mAb therapy may also increase efficacy of radiotherapy and other chemotherapeutic agents
Session 104: Stem Cells
• Two major categories of cells in adults
o Cells that are formed to last a lifetime (ex. Auditory hair cells)
o Cells that are replaced
♣ By simple duplication: differentiated cells divide (ex. Differentiated hepatocytes in liver, beta cells in pancreas)
♣ From stem cells: to replace cells that undergo rapid turnover (ex. Blood, skin, intestine)
• Stem cells
o Cells that can reproduce themselves as well as generate specific types of more specialized cells
o Properties
♣ Can undergo endless asymmetric cell division
♣ No replicative senescence (telomerase continually expressed)
♣ Each daughter cell can remain a stem cell or commit to a pathway that leads to terminal differentiation
♣ Self-renewal is the ability for a cell to proliferate in the same state
♣ Note: Rb is always phorphorylated (remember??)
o Asymmetric division
♣ Not fully understood
♣ Can be environmental (due to different environment of daughter cells) or divisional (division of RNA, proteins is asymmetric between daughter cells)
• Progenitor cells (transit amplifying cells)
o Staged between stem cels and differentiated cells
o Also called transit amplifying cells since they usually divide while ‘transiting away’ from the stem cell niche
o Related to stem cells but do not have the unlimited capacity for self renewal
o Usually more differentiated than stem cells and have become committed to a particular cell type
o Use of progenitor cells keeps the number of stem cells low and slowly dividing
♣ Reduces the potential for genetic damage and cancer
o Example: Hematopoietic Stem Cells
• Stem cell ‘Potency’
o Totipotent: can form every cell type including the trophoblast cells of the placenta (zygote)
o Pluripotent: can form every cell type except trophoblasts (ESCs)
o Multipotent: can form a limited number of adult cell types
o Unipotent: can form only one cell type
• Normal stem cells grouped into:
o Embryonic (pluripotent) stem cells: capable of developing into all cell types of the body, isolated from inner cell mass of blastocysts
o ‘adult’ stem cells: involved in replacing and repairing tissues of a particular organ, can typically forms only a limited subset of cell types, multipotent or unipotent
• Adult Stem cells
o Many/most? Adult organs contains committed stem cells
o Difficult to identify, isolate, purify as they are rare (estimated .1-3% of cells)
o
Low rate of cell division, do not proliferate readily
o In use medically ex: bone marrow transplants are essentially stem cell transplants
o Tissues and organs that undergo continual renewal (skin, intestine, breast, blood, etc) contain stem cells in areas known as adult stem cell niches
♣ Allows for controlled stem cell proliferation, differentiation of progeny that leave niche
♣ Produce paracrine factors that regulate proliferation, prevent differentiation, when cells leave niche they begin differentiation
♣ Intestinal niche: Wnt signaling maintains stem cell niche
• Other sources of multipotent stem cells:
o Mesenchymal Stem cells (MSCs)
♣ Multipotent
♣ Found in several adult tissues: bone marrow, adipose, dental pulp, breast milk, intestine
♣ Able to give rise to numerous mesenchymal (stromal) cell types: bone, cartilage, muscle, fat
o Amniotic epithelial cells: from the amniotic membrane in human term placenta
♣ Do not express telomerase and therefore do not make teratomas after transplantation
♣ Express markers that are present on ESCs, can differentiate into 3 germ layer in vitro
♣ Not used extensively now but might prove to be a source of SCs for tissue regeneration
o Fetal stem cells: found in the organ of fetuses
♣ Are somewhat more differentiated than ESCs, generate a limited number of cell types
♣ Do not form teratomas in vitro
♣ Up to 12 weeks, cells have less chance of rejection than cells derived from umbilical cord, marrow
♣ Even more controversial than ESCs
o Umbilical cord stem cells: Derived from the umbilical cord epithelium and cord blood
♣ More primivitive subpopulation of mesenchymal stem cells than bone marrow, less likely to generate host response
• Embryonic stem cells
o All cell types can be generated (in theory) from ES cells
o Most differentiated cells express only 10-20% of genes, ESCs express 30-60%
o Thought to be due to accessible chromatin structure
o Low-level expression of many cell surface receptors, enabling them to respond to many signals
o DNA methylation pattern is critical
♣ DMRT1 remethylates DNA after it becomes demethylated in the zygote
♣ Remethylation is required for pluripotency
o ES cells rely on ‘master’ transcription factors
♣ Oct4, Sox2, Nanog activate genes encoding proteins and miRNAs for self-renewal and pluripotency and repress genes that induce specific differentiation pathways
• Possible use of ES cells therapeutically to restore or replace damage tissue research and controversy
o Undifferentiated ES cells can form teratomas (can contain hair, teeth, bone, eyes, limbs, etc), so ES cells must ALL be differentiation before implantation
o Primary experimental sources
♣ Therapeutic cloning (somatic cell nuclear transfer = SCNT)
• Involves replacing the genome of an oocyte with that of an adult cell
• Need an oocyte donor and a nuclear donor
• Remove egg remove spindle apparatus transfer nucleus into enucleated egg egg and cell fused with electric current culture embryo
• Ex: Dolly the sheep, BUT she died, thought that she was actually the chronologic age of the mother because of telomere shortening
• Advantages: reduces ethical concerns as doesn’t involve ESCs, no need to identify and clone genes to screen for traits of interest
• Disadvantages: very inefficient, clones have medical problems (bad epigenetic programming), human oocytes manipulated to SCNT don’t develop to blastocyst stage, some concerns about obtaining human oocytes
o One study was able to get to blastocyst stage by injecting fibroblast nucleus into haploid cell triploid cell
♣ Excess eggs from in vitro fertilization (IVF)
• Ethical debate regarding the use of ESCs from IVF embryos
• Pro: ESC research fulfills ethical obligation to alleviate human suffering, IVF embryos will be discarded anyway
• Against: ESCs are taken from a blastocyst that is then discarded (murder?), risk of commercial exploitation of participants, ESC research will lead to human cloning
Session 105: Stem Cells and Disease
• Current role of stem cell therapy in regenerative medicine
o Bone marrow, umbilical cord, and peripheral blood stem cells are the only SC therapies routinely available
♣ Bone marrow transplant has been used to treat leukemias, other blood disease for 30 years
• Recently being used experimentally for other diseases: epidermolysis bullosa
♣ Umbilical cord has a relatively high number of MSCs, less prone to rejection
♣ Peripheral blood SCs can be used instead of bone marrow, less invasive to obtain
• Potential role of stem cell therapy
o Repair of degenerating or lost tissues
♣ Repair of neurons for spinal injuries, cardiac muscles after MI, neurodegenerative diseases
o Gene therapy for diseases
♣ Current treatment for muscular dystrophy, Type 1 diabetes, leukemias and other hematopoietic disorders, but potential for treatment of many other diseases
• The ‘Selling’ of Stem Cells
o Illegal since 1984 to sell any body parts (National Organ Transplant Act)
♣ BUT does not apply to blood stem cells obtained by apheresis – used in 2/3 BMTs
♣ Problem for poor because of pressure to sell body parts (blood, plasma not covered)
o Why allow the sale of stem cells (or body parts)?
♣ Very difficult to obtain matches, especially for mixed race individuals
♣ Children with with leukemia and aplastic anemia are in desperate need of stem cells
• Induced pluropotent stem cells (iPCSs) – most prominent type of stem cell therapy
o
Can be ‘made’ from multipotent cells by forcing the expression of certain transcription factors
o First done in 2006 by Shinya Yamanaka (nobel prize in 2012) with cocktail of 4 transcription factors
♣ Transformed a mouse fibroblast into a cell that appeared identical to an ES cell
♣ Transcription factors c-Myc, Klf4, Oct-3/4, Sox2
• Klf4 binds to beta-catenin, activates telomerase gene
• Oct-3/4 induce Sox2, which induces Nanog and Fbx15
o Note: dedifferentiation and regeneration has been known for some time in lower organisms
♣ Ex: ‘immortal jellyfish’ dedifferentiates back into an amoeba-like blob and then regenerates back into a jellyfish
o Testing for pluripotency
♣ When aggregated together, cells form a teratoma: tumor-like structure with all 3 germ layers
♣ Transcription and DNA methylation pattern were found to be almost identical to that of normal mouse ESCs (the concordance between the two varies with the cells and the labs)
o Curing a human disease in mouse using ePSCs
♣ Humanized sickle cell anemia mouse model
• Harvest tail tip fibroblasts infect with Oct4, Sox2, Klf4, c-Myc viruses correct sickle-cell mutation in iPS cells by specific gene targeting differentiate into embryoid bodies transplant corrected hematopoetic precursors back into irradiated mice
♣ In vivo reprogramming of adult pancreatic exocrine cells to beta-cells
• NGN3, Pdx1, Mafa transcription factors put into viral vector
• Injected vector into exocrine pancreas cells ‘exocrine’ pancrease cells produce insulin like beta-cells and corrected diabetic phenotype!
o Technical considerations for making iPSCs
♣ Choice of factors, methods of factor delivery, choice of cell type, parameters of factor expression, derivation of conditions, identifications of iPSC colonies, expansion and characterization
♣ Only about 1% of the cells de-differentiate completely, making it rather inefficient
♣ Regulation of expression is key – difficult to regulate post-translationally regulated events
o Uses of iPSCs – huge potential in medicine!
♣ Cell/organ therapies
♣ Disease modeling – neurological diseases, cardiovascular modeling, hepatic
• Can use diseased iPSCs to study disease (better than mouse models)
♣ Drug development – especially for mixed races and races not commonly tested
♣ Regenerative medicine
• Can generate early stem cells that have the exact genotype of the patient
• Can theoretically correct genetic diseases
• Minimal ethical issues
• Propensity to be tumorigenic (readily forms teratomas as with ESCs)
• Likely to be more tumorigenic because of use of viral techniques for DNA insertion
• Throughput is low; only a few cells are ‘induced’
• Cancer stem cells (CSCs)
o Some cancer may be considered a disease of stem cell regulation
♣ Evidence is accumulating that indicates that tumors can arise from adult stem cells
♣ Normal stem cells have a hierarchy of slowly dividing cells producing more differentiated cells
♣ Cancer cells seem to be organized in the same way
• Cancers of skin, intestine, blood are very frequent, yet the only cells that are around long enough to accumulate enough mutations are the adult stem cells
o Thought that a stem cell may undergo oncogenic transformation, lose important homeostatic control mechanisms
o Cancer stem cells may be responsible for cancer recurrence in some cases
♣ CSCs appear to repopulate cancers through self-renewal and differentiation of all the tumor cell types
♣ Origin of these CSCs is unclear
o Few cells in tumors are tumorigenic
♣ <1% chance that random tumor cell will generate a new tumor when transferred
♣ Thought that few tumorigenic cells are cancer stem cells
o Cancer Therapies
♣
Current therapies may promote CSC survival and propagation
• Radiation and many chemotherapies target rapidly dividing cells, BUT CSCs would replicate infrequently Thought therapies kill off the bulk of the tumor but NOT the cancer stem cells
• SCs and CSCs are naturally resistant to chemo agents as they have ABC transporters that pump out the drugs (so that the stem cells won’t accumulate mutations) only the susceptible cells die and the resistant cells live
o Ex: paclitaxen in ovarian cancer
o
Prevailing hypothesis is that recurrent cancers are largely the consequence of CSCs slowly repopulating the area
Session 106: Genetic Modifications in Medicine
• Genetically Modified Organisms (GMOs)
o Genome of an organism is modified by genetic engineering techniques
o Routinely used:
♣ Pharmaceutically important drugs (insulin, growth hormone)
♣ Agriculture to enhance the resistance, storage, taste, amount of food products
♣ Environment (clean up spills, ‘sterile’ mosquitoes)
♣ Research purposes
♣ Fun – GloFish, GFP Axolotls!
• Transgenic Mice
o Have ‘foreign’ gene introduced into their genomes – knockin
o Used to study ‘normal’ gene function in mammals and to model human diseases
o The inserted DNA (transgene) usually confers gain of function of that gene
♣ Random insertion of gene, doesn’t typically disrupt genes
o Typically done by pronuclear injection
o Gene is inserted into an Expression vector, typically a bacterial plasmid
♣ Requires promoter, multiple cloning site (MSC) with restriction sites where gene can be inserted, region that encodes a peptide that can be recognized by an antibody, resistance genes for screening purposes (such as ampicillin resistance marker for bacteria)
o Pronuclear injection
♣ Expression vector injected into male pronucleus via non-homologous recombination, then egg is transferred into foster mother
♣ About 10-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line, then can breed mice
o Advantages:
♣ Quick and easy, transgene usually not lethal so some phenotype will be observed
♣ Used extensively for GMOs
o Disadvantages:
♣ Tegulation of the transgene is usually not normal, there can be ‘dosage’ effects because of the multiple gene copies
♣ Endogenous gene is still active so looking for an effect ‘on top’ of the endogenous gene
♣ Transgene protein must be identified separately from the endogenous protein
• Null Mice
o A knockout is the germ line deletion of a specific gene
o Advantages:
♣ Very useful for exploring functions of genes as it assesses what happens in an organism when a gene is missing
♣ Very useful to study development
o Disadvantages:
♣ Interpretation can be complicated by ‘compensatory’ increases in other genes
♣ Deletion may be lethal, slow and a lot of work (and very expensive!)
o Creating gene knockouts in mice
♣ Gene introduced by homologous recombination such that the endogenous gene is removed and a new one is inserted – done is ES cells growing in culture
♣ ES colony with knock out injected into early embryo, which is then implanted into foster mother
♣ Breed heterozygous offspring to get the null animals and hope its not lethal
o Creation of ‘conditonal’ knockouts
♣ Gene can be disrupted only in a specific tissue or at a specific time in development
♣ Most commonly uses the Cre-Lox system
•
Target gene replaced by the same gene that is flanked by the LoxP sites, which are recognized by the Cre recombinase recombinase enzyme then does site-specific recombination using the LoxP sites
• Lox mouse in then mated with a Cre mouse that has the recombinase under the control of a tissue-specific promoter or one that can be induced
• Ex: Estrogen receptor that is sensitive to tamoxifen, the Cre-Lox mouse can be injected with tamoxifen at any time, recombination is induced to remove the targeted gene, effects of the loss can be determined
• Human gene therapy
o Requires the same site-specific knockin and knockout technologies or analogous methods
o Germline human gene therapy – illegal at this time
o Somatic human gene therapy: therapeutic genes are transferred into the somatic cells of a patient
♣ Becoming increasing useful, but not fulfilled expectations
♣ Problems limiting somatic gene therapy
• Vectors often have viral particles, leading to potential problems with toxicity, immune and inflammatory responses
• Corrections are often transient (gene methylation?)
• Chances of inducing tumors
• Regulation and delivery are difficult
• Cloning
o A ‘clone’ is a set of individuals that are genetically identical because they descended from a common ancestor
o Human cloning refers to making an identical copy of a human individual
• Human reproductive vs. therapeutic cloning
o Reproductive cloning (SCNT): embryo generated is implanted into the uterus of a foster mother to create a new individual (could also use iPSCs to get embryo) human cloning
♣ Only require the genome of one individual, a form of asexual reproduction
♣ Currently unsafe with about 95% of cloning attempts ending in miscarriages, stillbirths, etc
♣ Cloned individuals are often biologically damaged
♣ In the future, could replace of cherished loved one OR provide children for those who are sterile or homosexual
o Therapeutic cloning (SCNT): embryo generated is used as a source of ESCs cell or tissue regeneration
Session 107: Intro to Development and Disease
• Basic components of development
o Increase is number of cells and size of organism
o Increase in complexity with diverse cell types
o Patterning (blueprint) and morphogenesis (construction)
o Still occurs after birth
o Influenced by both genes and the environment
• Animal models used to study development (for ethical reasons)
o Experimental models (develop outside mother) – xenopus, chick
o Gentic models: mouse and zebra fish (vertebrates), c elegans and drosophila (invertebrates)
o Much of what we know about development is universal
♣ 3 common germ layers: endoderm, mesoderm, ectoderm
♣ 50% of human genes are conserved in c elegans and drosophila
• Essential genes in embryonic development (first found in drosophila)
• Pair rule genes are transcription factors
o Vertebrate Pax genes – expressed in segments
♣ Mutant Pax1: undulated mouse with shortened vertebral column
♣ Pax3 homozygous mutants (splotch mouse) show spina bifida, brain/neural crest defects
♣ Pax3 heterozygous similar to splotch mouse, pigment defects
♣
Waardenburg’s syndrome patients have mutations in the human homologue of the Pax3 paired box gene allowed sequencing of human Pax 3 analog
♣ Synteny: conserved organization of human and mouse chromosomes
• Regulating expression of different genes in time and space gives rise to diverse cells types
o Regulated expression at transcriptional, post-transcriptional level, or post-translational level
♣ Transcriptional – transcription factors (enhancers, etc), chromatin remodeling
♣ Post-transcriptional level – RNA processing, inhibitory proteins, miRNA
♣ Post-translational level – phoshorylation state, nuclear vs. cytoplasmic
o Regulation of master transcription factors and hox transcription factors
• Master transcription factors – Cell specification/fate
o Cell face specification: Ex. Fat cell vs. muscle cell
o Pax-6
♣ In drosophila, artificial expression in leg gives ectopic eye on leg
♣ Can induce undifferentiated cells into an eye, required for eye formation
♣
In humans, aniridia is caused by mutation in the pax-6 gene (black iris, poor vision)
♣ Transcription factor, contains conserved motif for DNA binding (pax-1, pax-3, pax-6)
• Paired box and paired homeobox
• Mutation in these causes impaired function – required for DNA binding
o myoD – master transcription factor for muscle
♣ Fibroblast myoD muscle cell
• Hox genes – specification of cell identity (brain vs. spinal cord, what KIND of neuron) along anterior-posterior axis
o Transcription factors that have a homeobox DNA binding region
o Found in a cluster along the same region of the chromosome
o Position of the genes in the homeotic complex corresponds to body segment
o Mutations in Hox genes cause homeotic transformation
o
Humans have multiple redundant copies of Hox genes, but can still have homeotic transformations
♣ Ex: Hoxb-2 expressed from shoulder to legs, Hoxb-4 expressed from arms down
♣ Hoxb-4 knockout mouse: C2 vertebra transformed into more anterior C1
o Hox genes and human disease
♣ HoxD13: synpolydactyly – short, fused fingers
o Retinoic Acid is a teratogen – caused misexpression of hox genes
♣ Causes homeotic transformation in the hind brain
♣ Causes more anterior expression of Hoxb-1 in hind brain rhomboid 2/3 intro rhomboid 4/5
o How is A/P pattern of Hox gene expression set up?
♣ miRNAs play an important role in where Hox genes are expressed
• miRNA-10 targets Hoxb4, miRNA-196 targets Hoxb8
• Wherever these miRNAs are expressed, you get targeted destruction, silencing
Session 108: Spatial and Temporal Signaling in Development
• Induction – inducer, responder, competence
o A process by which one population of cells (inducer) affects the development of another (responder) through signaling
o Two types
♣ Paracrine – involve diffusible molecules
♣ Juxtacrine – involve cell contact
o Example: Eye development
♣ Normal induction of lens by optic vesicle (inducer)
♣ Only ectoderm in head region is competent to receive inducer signal and respond
♣ Optic vesicle has high expression levels of FGF8 inducing factor
♣ Head ectoderm expresses pax-6, required for ectoderm to be competent for lens induction
• Most embryonic inductions are mediated by secreted signaling factors
o Can act on remote cells in paracrine fashion
o Can form gradient, affecting cells differently depending on the concentration of the signaling factors
o Always activate intracellular signaling pathways
•
Four key signaling pathways in development - involved in INDUCTION
o FGF pathway – binds to tyrosine kinase receptor, activates MAP kinase pathway; mutations typically affect bone development
♣ BMP7: kidney and eye development, skeletal patterning
♣ BMP2: heart development
♣ BMP8: spermatogenesis
o Hedgehog (Hh) – membrane receptor Patched stops inhibition of Smoothened Gii enters nucleus transcription, Gli is either an activator or a repressor
o Wnt pathway – Frizzled Disheveled inhibits GSK-3 b-catenin not degraded
o TGFb – Smads (see previous)
♣
Associated with diseases of limb formation
• Sequential inductive interactions lead to pattern formation
o Specification of embryonic axis
♣ A/P axis
♣ D/V axis
♣ L/R axis
o Eye development
♣ Optic vesicle induces formation of optic placode lens placode induces formation of optic cup lens capsule induces formation of cornea
o Neural Tube Patterning – dorsal/ventral patterning
♣
Two signaling pathways – sonic hedgehog (notocord) and BMPs (ectoderm)
♣ Ventral patterning of neural tube induced by Shh secreted by notocord (HEDGEHOG signaling pathway) – D/V concentration gradient
• High concentration in floor plate motor neurons
• Lower concentration towards roof plate different types of interneurons
♣ Dorsal patterning of neural tube induced by BMP4 and 7 secreted by epidermis and roof plate turns on TGFb signaling pathway
• High concetration in roof
♣ Gradients of the two paracrine factors on opposite ends of D/V axis results in production of different transcription factors, which specify different neuronal cell fates
• Hedgehog signal transduction pathway mutations
o Gli truncation leads to Pallister-Hall syndrome
♣ Gli is continually active as a repressor
♣ Lots of digits – no proper patterning
o Greig cephalopolysyndactylyl is due to loss of function mutation of Gli
♣ Gli cannot act as an activator or a repressor
♣ Megalocephaly, broad thumb, duplicated big toe – duplicated fused digits
o Mutations in patched receptor lead to Gorlin’s syndrome/basal cell syndrome
♣ Overactivation of Hh signaling due to constitually active Smoothened signaling even without hedgehog ligand (Patched normally inhibits Smoothened in absense of Shh ligand)
♣ Cancer due to overproliferation of cells in skin, eye
o Loss of hedgehog molecule leads to holoprosencephaly cyclopia
♣ Major developmental disorder
♣ Affects midline patterning
♣ Can be as mild as only having a single front tooth to having a single eye (cyclopia)
♣ Can also be caused by defects in the synthesis of cholesterol
• Hh requires cholesterol modification to create smooth Hh gradient through signal diffusion
• Jervine alkaloid inhibits cholesterol synthesis causes similar symptoms
• FGF signaling pathway mutations – generally affect bone formation and limb development
o Crouson’s, Pfeiffer, and Apert Syndromes
o Achondroplasia – congenital dwarfism
• Wnt Signaling and Disease – generally gives rise to cancer
o DES (diethylstilbestrol) – teratogen causing abnormal reproductive tract in fetus
♣
DES inhibits production of Wnt7a in epithelial tissue
♣ Loss of Wnt7a signaling means Hox and Wnt5a are not induced in mesenchyme
o APC mutations lead to FAP hereditary colon cancer
Session 109: Mechanics of Morphogenesis and Cell Adhesions
• Morphogenesis
o How are tissues formed from populations, how do they migrate to the correct layer, etc…
• Three germs layers
o Holfreter’s experiment to show cells sort themselves
o Reconstruction of dissociated skinc ells from 15 day mouse embryos
• Cell adhesion as a mechanism of morphogenesis
o Generates boundaries between different cell types
o Formation of tissues from individual cells, and maintenance of tissue integrity
o Formation of organs and maintenance of organ integrity
o Establish connections between different cell types that need to communicate (neurons and mucle)
•
Cell adhesion molecules
o Cadherins – large family (desmosomes, adherent junctions)
♣ Classic cadherins: E-cadherin, P-carherin, N-cadherin
♣ Homophilic adhesion
♣ Injection of cadherin mRNA in xenopus embryo results in loss of cell adhesion
♣ N-cardherin establishes boundary between neural and epidermal ectoderm
♣ To form adhesion, Ca2+ must be in solution and must be associate with actin cytoskeleton
• Associated with cytoskeleton by catenins (remember Wnt pathway???)
♣ Signaling pathways and disease (crosstalk)
• Wnt signaling pathway b-catenin: colon carcinomas, melanomas probably due to loss of adhesion, contact inhibition through crosstalk with Wnt signaling pathway
♣ Diseases associated with cadherins – normally loss of classical cadherins leads to death of embryo due to importance in embryogenesis
• E-cadherin: tumor malignancy (loss of contact inhibition cell proliferation)
• Demosomal cadherin: pemphigus vulgaris (autoimmune disease, Ab made to desmonsomal cadherin)
o Skin, mucous membrane blistering due to loss of desmosomes in stratum spinosum layer of skin
• Cadherin 23, protocadherin 15: usher syndrome
o Common form of hereditary deafness – disorganization of hair cells due to loss of stereocilia connection by cadherin molecules
o Retinitis pigmentosa
o IgCAMs – very large family
♣ Calcium independent
♣ Extracellular globular domains held together by disulfide bonds
♣ Mediate homophilic and heterophilic binding with other molecules, including extracellular matrix (collagen, proteoglycans, fibronectin, laminin)
♣ Weaker adhesions than cadherins
♣ Generally thought to be of importance for transient cell adhesion
♣ Generally found in neuronal cells (NCAM) important in axon guidance and neurite growth
♣ Associated Diseases
• CRASH and L1 (IgCAM linked to actin cytoskeleton by ankyrin)
o Corpus callosum hypoplasia
o Retardation, mental
o
Adducted thumbs
o Spastic paraplegia
o Hydrocephalus
• Autism, Schizophrenia, Cancers
o Integrins – large family (hemidesmosomes)
♣ Heterodimeric proteins composed of one alpha and one beta subunit
♣ Cell-cell (typically heterodimeric) and cell-extracellular matrix (ECM) adhesion
• Links EXTRACELLULAR matrix (via talin, viculin, alpha-actinin) and INTRACELLULAR actin cytokeleton
♣ Dependent of extracellular divalent cations (Ca, Mg) for ECM binding
♣ Much weaker than cadherins, but typically present in high concentrations (like Velcro)
♣ Important in cell migration
♣ Associated Diseases
• Angiogenesis, Inflammatory diseases, cancer, myopathy
• Epidermolysis Bullosa
o Skin blister disease, may be fatal
o Defect in cell adhesion, but defect in hemidesmosomes in basal layer of skin that connect cells to basement membrane
♣ Play important role in cell migration – migration of neural crest cels, migration of blood cell precursors to liver, bone marrow, etc…
• Cell Adhesion are important
o Tissue boundaries, cell sorting
o Epithelial adherens junctions maintain tight protective layer
o Cell migration, neural crest cells must migrate, requires cell adhesion molecules
o Axon guidance, synapse targeting, and adherens (connection between neurons, muscle cells)
• Types of cell adhesion
o Homophilic binding – one adhesion molecule binds to same molecule on other cell
o Heterophilic binding – adhesion molecule binds to different molecule on other cell
• Cell migration as a mechanism of morphogenesis – extension, attachment, translocation, de-adhesion
o Migration of cells during gastrulation
♣ Migrating ectoderm forms endoderm and mesoderm
o Neural crest cells undergo extensive migration
♣ Neuroectoderm cells that for neurons, Schwann cells, pigment cells
♣ Defects can give piebaldism, where pigment is missing from forehead, stomach
♣ Hirschspring’s Disease – congenital constipation of lower bowels caused by absence of ganglia that regulate preristalsis
o How cells migrate
♣ Cells extent filopodia, lamellipodia and form contacts with substratum
♣ This connected in mediated by integrins
• Helps create force/traction so cells can move against substratum
o How do cells decide where to migrate?
♣ Haplotaxis: migration based on changes in adhesiveness of the substratum
♣ Specific substrate guidance: migration along a pathway made of a specific substance (eg laminin, collagen) – sensory neuron grows processes onto laminin but not collagen
♣ Chemotaxis: migration regulated by a gradient of diffusible substances sensed by the cell via cell surface receptors (like bread crumbs) – neutrophil migration
o Example: directing axon migration in neural tube
♣ Floorplate neurons must migrate ventrally, then anteriorally
o Chemotactic cues can be attractive and/or repulsive
♣ Attractive: N-formylated peptides produced by bacteria attract neutrophils which sense cue via a cell surface receptor
♣ Repulsive: Slit repels axons expressing the Slit receptor, Roundabout (Robo)
• Slit found at midline, so axons/neurons expressing Robo won’t be seen at midline
• Slit knockout axons with Robo found at midline
♣ Both: Netrins are secreted by floorplate on the central part of neural tube, direction the ventral migration of some axons and repelling migration of others
• Some neurons are attracted to Netrins, some are repelled
• Why? Different neurons express different receptors type of guidance molecule and receptor
Session 110: Human Malformations and Teratogenesis
• General
o Typically occur in the first 12 weeks of gestation – period of organogenesis
o Birth defects are common: 1/30 babies are born with a ‘birth defect’
o 5th leading cause of death in children 5-14 years
o 4th leading cause of infant mortality worldwide
o Causes
♣ 95% genetic – complex of multifactorial 50%
♣ 5% environment – infections, prescription drugs, recreational drugs/ethanol, method of conception
• Genetic Malformations
o Chromosomal basis of birth defects
♣ Standard chromosome studies will be abnormal in about 4% of infants with birth defects
♣ With array CGH up to 20% will be identified to have a significant chromosome abnormality
o Single Gene Disorders
♣ Although there are many single gene disorders that cause birth defects, there are no current means for screening multiple genes at once
♣ Testing relies of ability of clinician to recognize pattern of malformations – syndrome
♣ Syndrome Recognition
• Clinical recognition of a constellation of findings that when identified together in a patient constitute a known condition
• We do not known the exact cause for the majority of birth defects - Multifactorial
o The ‘complex’ or ‘multifactorial’ model is used to explain the majority of birth defects
o Most common defects – congenital heart defects, cleft lip and palate, neural tube defects
o Neural tube defects
♣ 1930’s – noted that women with good nutrition had lower risk
♣ 1980’s – found folic acid was protective, confirmed in early 90’s through case control studies
♣ Folic acid is now given prenatally to women to prevent NTD
♣ Cereals and grains are now fortified with folic acid
♣ Rates have decreased since fortification with folic acid
♣ Hypothesis: since folic acid lowers the risk of NTD, then the genetic cause may lie in genes that encode proteins or enzymes that effect folate metabolism
• Found that MTHFR polymorphism T/T homozygotes (10% of population) increase risk – homozygous mothers have relative risk of 1.6
♣ May be gene/ environment interactions
o Cleft lip and palate
♣ Like NTD, there is an increased risk of cleft lip in palate in subsequent pregnancies (5-7% risk of recurrence)
♣ Complex inheritance model
♣ Multiple studies have shown an increased risk of cleft lip/palate to mothers who smoke – odds ratio of 1.3
• Environmental causes of birth defects - teratogens
o Timing
♣ There are critical periods in embryonic periods where embryo is most susceptible
o Alcohol – 30-60% fetuses affected
♣ Recognizable pattern of facial findings (short nose, flattened midface, thin upper lip, etc), growth delay, heart defects, cognitive disability and behavioral difficulties
♣ Later exposure results in cognitive disability and behavioral difficulties without facial findings
♣ There is no known safe amount of alcohol during pregnancy
♣ Annual costs related to caring for ethanol exposed infannts is in the BILLIONS
o Prescription medications
♣ Isotretinoin – 30%
♣ Thalidomide – 10%
• Associated with severe malformations of their arms and legs
• Thalidomide was used as a sedative to help pregnant women remain ‘calm’
♣ Warfarin – 8%
♣ Diazepam – 1%
o Maternal Infections
♣ Cytomegalovirus (CMV)
• 1% of infants are infected with CMV at birth
• 10% of infected infants can have sequelae
o Hearing loss, microcephaly, brain malformations, rash
♣ Rubella
• Common prior to universal immunization in the 1960s
• Concerns with re-emergence in area with low immunization compliance
• 90% of women infected during the first trimester will have infected offspring
• Clinical Findings
o Cataracts, deafness, brain malformations, microcephaly, rash (blueberry muffin baby)
o Assisted Reproduction
♣ 10% of couples experience infertility
♣ Now ask mode of conception as part of history taking in genetics clinic
♣ In vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSE)
• Higher incidence of imprinting disorders (especially maternal imprinting): Angelman, Beckwith-Wiedmann
• Higher incidence of cleft lip/palate, hypospadias, esophageal atresia, imperforate anus
Session 111: Apoptosis in Development
• Apoptosis is genetically programmed
o Apoptosis is programmed cell death – genetically controlled
♣ Cell membrane is intact, no cytoplasm leaks out
♣ Genes are turned on to induce morphological changes – degradation of cell contents
♣ Chromatin compaction gene causes DNA condensation and cleavage
o Necrosis is cells that are damaged by injury or exposure to toxic chemical, cells swell, lyse, cause inflammation of surrounding tissues
• Apoptosis in tissue morphogenesis
o Sculpting tissue/organ
♣ Fingers, limb development
♣ Tube formation (hallowing out)
♣ Bone formation – hypertrophy and apoptosis of chondrocytes to for bone
o Deletion of unwanted structures
♣ Vestigial structures removed – like tadpole’s tail
♣ Tissue homeostasis – mammary secretory epithelial cells that increase during lactation die after weaning
♣ Development of male and female reproductive organs (Mullerian and Wolffian duct)
o Regulation of number of cells
♣ Number of motor neurons are determined by size of target tissue to be innervated, neurons that don’t ‘make it’ undergo apoptosis
o Elimination of damaged or harmful death
♣ 95% of thymocytes generated in thymus (self-reactive) die
♣ Cells that have incurred DNA damage
o Production of specialized cells like lens epithelial cells, keratinocytes, RBCs
♣ Lens cells are actually dead – apoptosis of nucleus occurs but cell is not phagocytosed
•
Caspase cascade
• Apoptosome
• Suicide (intrinsic) pathway via mitochondria
o First discovered in c elegans
o Induction of procaspase activation
o Caspase-9 – protease, induces caspase-3 (caspase cascade)
o Caspase Cascade:
♣ Release of cyt c binds to Apaf1 causes conformational change forms apoptosome recruitment, cleavage of procaspase-9 active caspase activates other caspases (caspase-3) cleavage of cytosolic proteins, nuclear lamins, etc…
o Bcl2 proteins
♣
Anti-apoptotic proteins (Bcl2, Bcl-Xl) – 4 BH domains
• Binds to pro-apoptotic BH123 proteins to block pore formation cyt c cannot escape for mitochondria
♣
Pro-apoptotic BH123 proteins – 3 BH domains (Bax)
• Found in mitochondrial membrane
• Aggregation causes formation of pores to release cyt c caspase cascade
♣ Pro-apoptotic BH3-only protein – 1 BH domain (Bid, Bad?) – sequester Bcl2 away from Bax channel
o Expression of Bcl-2 is dependent on the Mift transcription factor
♣ Mift is activated by MAP kinase cascade transcribes genes that ensures cell survival of melanocytes
♣ Piebaldism results in loss of melanocytes regulated by the Mift transctiption factor
o Bcl-Xl expression controlled by erythropoietin
♣ Bcl-X regulates how many red blood cells go into circulation
♣ Binding of erythropoietin to receptor causes downstream transcription of Bcl-Xl anti-apoptotic protein more RBCs survive
• Signals inducing apoptosis
o Loss of trophic factors
♣ In presence of survival factors, Bcl2 production is increase apoptosis blocked; inactivation of pro-apoptotic BH3-only Bcls protein
♣ Absense of survival/trophic factors promote apoptosis
• Activation of caspase cascade
o Damaged cells via p53
♣ PUMA (p53 Upregulated Modulator of Apoptosis)
• Pro-apoptotic Bcl2 protein – contains BH3 domain, binds to anti-apoptotic Bcl2 proteins
• Murder (extrinsic) pathway receptor mediated
o Generally induced by cytotoxic T cells
o T-cell receptor binds to histocompatability molecule produced FasL ligand binds to Fas receptor
on target cell (ALL cells express Fas receptor)
o Causes trimerization of receptors death domains aggregate (allows formation of DISC) recruits and actvates caspase 8 more caspases recruited (caspase 3)
o Mutations in FasL or FasR or caspases in pathway, have defective apoptosis
o FasL and immune privileged sites
♣ Very little immune response in brain, eye, etc
♣ Have physical structures AND immune blockers
♣ Eye cells have FasL that is constitually active induces apoptosis in immune cells that enter the eye
♣ In these tissues, immune response (inflammation) would cause a lot of damage
• Homeostasis – balance between cell division and cell death
o Too much cell proliferation causes cancer, SLE, rheumatoid arthritis, polycythemie
o Too much cell death causes neurodegenerative diseases (huntington’s, ALS), autoimmune diseases (AIDS), stroke, MI
Session 112: Organogenesis
• How the endoderm gives rise to the digestive and respiratory tracts
o Tissue becomes specified to a certain organ, then cell differentiation occurs
• Gut development
o
Differentiation into foregut, midgut, hindgut is caused by relative levels of Wnt, Fgf4, BMP signaling molecules
o Different parts of gut express different transcription factors (see figure)
o Know Pdx1 required for pancreas formation
o As gut invaginates, Sonic Hedgehog (Shh) is expressed in hindgut first turns on expression of specific Hox genes
o Paracrine signals from cardiogenic mesoderm (FGF)
♣ Highest levels received by lungs, then liver
o Liver formation
♣ Receives FGF signal from cariogenic mesoderm
♣ Liver endoderm made competent to receive signal by BMPs
♣ Induces formation of hepatocytes, begins expressing Prox1 allows cell proliferation
• Reduces levels of E-cadherin
• Proliferating cells need space, start proliferating into mesenchyme (which produces hepatocyte growth factors)
• Prox1 mouse mutant: no cell proliferation, no cell migration, high levels of E-cad
o Pancreas formation
♣ 2 buds formed one next to liver (ventral bud), one dorsal bud
♣ Eventually migrate and fuse
♣ Ventral endoderm
• Low FGF, BMP, but does express PDX1 (master transcription factor for pancreas)
• Migrates to dorsal bud, where they fuse
♣ Dorsal endoderm
• Also expresses PDX1
• Signaling from notocord, expresses FGF2 and activin
• Induces endoderm to turn down Shh induces pancreas formation
o Forcing Shh expression means dorsal pancreas doesn’t form
♣ Pdx1 transcription factor
• Master transcription factor
• Induces budding from the gut epithelium
• Represses gene expression characteristic of gut tube other than pancreas region
• Maintains repression of Shh in pancreas region of gut
♣ Pdx1 and disease
• Loss of Pdx1 results in loss of pancreas formation
• Humans homozygous for Pdx1 mutation do NOT develop a pancreas die
• Patients heterozygous for Pdx1 mutation develop MODY4 diabetes (Pdx1 is also important in beta cell formation)
• Lung formation
o Tbx4 helps tracheoesophogeal folds to fuse
♣ Loss of tbx4 leads to tracheoesophogeal fistula
o Mesenchymal interactions required for bronchial branching
♣ Fgf10 found in mesenchymal cells acts as a chemoattractant for lung epitherium
♣
Tips of lung buds express Fgf receptor that responds to signal migrate and grow towards cells expressing Fgf10
♣ Lung bug epithelium exposed to Fgf10 expresses sonic hedgehog (Shh) this signal diffuses to mesenchymal cells, which stop Fgf10 production
♣ This leads to BRANCHING
• Epithelium-mesenchymal interaction for organ fine tuning (found in many different tissues)
o
All organs have an epithelium (one of three germ layers) and mesenchyme (mesoderm or neural crest)
o Epithelial-mesenchymal inductions display regional specificity; mesenchyme specifies the structure to be formed
♣ In chick embryo, mesenchyme from wing, thigh, and foot transplanted with wing epidermal epithelium induces formation of wing feather, thigh feather, or scales, respectively
♣ Epithelium is a ‘blank slate’ induced to form different structures by mesenchyme
Session 113, 115: Limb Development
• Limb development is the most well understood organ development
o Malformed limbs are not lethal, unlike malformation of many other organs
o
Chick wing development is very similar to human limb development
• Initiation (where limb growth starts)
o What determines where limbs are formed? Hox genes (important in patterning along A/P axis)
♣ Hox genes specify region of the embryo, then master transcription factors are turned on
♣ Most anterior region where HoxC-6 is not expressed is where forelimb develops
♣ Most posterior region where there is no HoxC-8 expressed is where hindlimb develops
o
What induces limb bud formation?
♣ High expression of FGF10 in mesoderm where limb bud develops
♣ First expressed uniformly, then localized to where limbs develop (probably based on Hox genes)
♣ Induces epithelium (apical ectodermal ridge) to express Wnt3a FGF8 signals back to mesoderm to keep expressing FGF10
♣ Ectopic FGF10 can induce additional limb formation
o What specifies fore vs. hindlimbs?
♣ Tbx5 (forelimb) and Tbx4 (hindlimb) are important transcription factors
♣ Ectopic FGF10 between fore and hindlimb induces formation of chimera limb that expresses both Tbx5 and Tbx4
♣ Clinical: Holt-Oram syndrome
• Patients are heterozygous for mutations in Tbx5
• Also known as heart-hand syndrome
• Heart and upper limb malformations: absent thumbs, distally placed, duplicated, or triphalangeal thumbs
• Partial or total absence of forearm
• Proximodistal axis: FGF pathway
o Mesenchymal cells hold a lot of information, epithelium is a ‘blank canvas’
♣ ‘Progress zone’ (regional of mesenchymal cells) that undergoes proliferation
♣ Forms all cells of limb
♣ Apical ectodermal ridge responds to FGF signaling from ‘progress zone’ (see above)
• Produces Wnt3a and FGF8
• FGF8 is required to maintain cell proliferation in progress zone (also maintains FGF10 expression) but contains no positional information
♣ Mesenchyme induces and sustains AER and specifies type of limb; AER sustains outgrowth and development of limb and directs P/D growth
♣ AER only maintains proliferation of progress zone – implantation of old AER into young limb bud results in a normal limb (vice versa with young AER)
♣ Positional information lies with mesenchyme (progress zone) – implantation of old limb bud onto young limb bud results in only distal structures
o
How does mesenchyme specify the proximal-distal axis?
♣ Progress zone model: time - how much time is spent going through cell division determines what kind of structure develops (proximal vs distal)
♣ Early allocation and progenitor expansion model: space – there are four regions of limb bud, each region just expands in size as limb grows (cell fate is specified from the beginning to become proximal vs distal stuctures)
o Hox (A and D) genes specify the P/D regions of the limb (cell fate)
♣ Mutations in HoxD13 give mutations in most distal part of arm (digit formation)
♣
Deletion removing HoxD cluster leads to severe developmental defects in distal regions (11, 12, 13 ulna, metacarpals, digits)
•
Anteroposterior axis: SHH pathway
o Anterior = thumb; posterior = pinky
o Zone of polarizing activity (ZPA) – region of the limb mesenchyme in the posterior limb bud
♣ Required for A/P patterning of the limb
♣ Secretes sonic hedgehog (SHH) (induced by FGF8 secreted by AER)
• Increases activator Gli3:repressor Gli3 ratio with the highest levels of activator in the posterior
• Initiates and sustains a gradient of BMP (2, 7 - TGFb family) signals in interdigital mesoderm specifies digit identity
• BMPs induced in webbing between digits specify digit identity
o Clinical: SHH misexpression and congenital limb defect
♣ SHH contains enhancer far away (in different gene); important for direction expression of SHH
♣ Mutations in enhancer lead to limb malformation extra digits, mirror imaged limbs
♣ Gli-3: Grieg cephalopolysyndactyly and Pallister-Hall syndrome with polydactyl, abnormal facial, cranial formation
• Dorsoventral axis: Wnt pathway
o Dependent on surface ectoderm
o Wnt7a is expressed in the dorsal but not ventral limb ectoderm
o 180 degree rotation of ectoderm partially reverses polarity on distal structures
o Lmx1 transcription factor, activated by Wnt7a
♣ Specifies dorsal identity in limbs
♣ Expressed only in the dorsal limb mesenchyme
♣ Ectopic expression of Lmx1 in ventral limb mesenchyme induces dorsal phenotype
♣ Lmx1 mutant mouse knockout results in mice with no dorsal limb
♣ Clinical: Nail-patella syndrome – dominant disorder resulting from mutations in Lmx1 gene
• Dorsal tissues are partially ventralized
•
Coordinating three axis
o Fgf8 is required to induce Shh expression, thus coupling A/P patterning to P/D growth
o Wnt7a is required to maintain Shh expression, coupling A/P patterning to D/V
♣ Knocking out Wnt results in abnormalities of D/V patterning and results in loss of 5th digit as well
o AER forms in response to FGF10 only where dorsal and ventral ectoderm are juxtaposed, coupling P/D growth to D/V patterning
• Morphogenesis – adhesion, apoptosis, migration; occurs after patterning, limb bud formation
o Sculpting the autopod (hand)
♣ Cell death is required
♣ Apoptosis occurs in intidigital regions, as well as space between radius and ulna
♣
BMPs are important for inducing cell death
• Inhibition of BMPs (by noggin) maintains interdigital tissue
• Also involved in inducing chondrocyte formation
• Note: BMP turns on FGF pathway
♣ TGFb/BMP signaling and disease (loss of GDNF?)
• Symphalangism – loss of second phalanges
• Brachydacyly – fusion of joints (no cartilage formation)
o Bone formation
♣ Bones must undergo apoptosis and chondrocyte formation
♣ Apoptosis shapes bone
• FGF and Disease
o Directs chondrocyte formation, and thus is important in bone formation
o Mutations give Pfeiffer syndrome (early closure of sutures, very severe), Crouzon syndrome, Apert Syndrome (includes syndactyly), achondroplasia (short limbs due to disturbed growth)
o Activation of FGF receptor is constitutive after mutation leads to p21 activation brings cells out of cell cycle (Cdk inhibitor) leads to cartilage growth stopping early stunting of growth
Session 116: Pharmacogenomics
• What happens to a drug when it enters the body?
o Absorption
o Distribution
o Metabolism
♣ Biochemical modification, xenobiotic metabolism often converts lipophilic chemicals to more readily excreted polar products (p450s)
♣ Metabolism can result in activation or deactivation of drug
o Excretion
o ADME process – each step is affected by genetic differences in people
♣
Receptors, ion channels, transport molecules, signaling pathways, metabolic pathways – all part of GENETIC PROGRAM of an individual
• Pharmacokinetics
o Time course of drug and metabolite levels in different fluids, tissues, extreta of body
o Different for different individuals
o Drug dose can be represented as the area under curve (AUC)
o Therapeutic window – amount of medication between the amount that gives the effective dose and the amount that gives more adverse effects (toxicities) than desired effeects
• Pharmacodynamics
o Biochemical and physiological disposition of the drug within the body (often related to receptor interactions and transport)
• Example: Cetuximab
o An EGFR inhibitor, given by IV for treatment of metastatic colorectal cancer, head/neck cancer
o About 75% of metastatic colorectal cancers have EGFR+
o BUT…40% do not respond to Cetuximab – have an activating mutation in the RAS gene
♣ EGFR signaling is not required to activate RAS pathway
o Genetic testing recommended to look for mutations in RAS before prescribing Cetuximab
• Pharmacogenomics
o Using what we know about genetic variations in individuals to predict drug response in an individual
o FDA currently has about 80 recommended companion genetic diagnostics with drugs
• Drug metabolism
o Phase I Enzymes: add or expose polar groups to inactivate enzymes or activate proenzymes by oxidation, reduction, hydrolysis
o Phase II Enzymes: conjugate other molecules to drug (methylation, sulphation, acetylation, glucuronidation) to increase mass of drug most often inactivate drug
• Drug dose and response is related to genetic variations
o Ex: Prozac overdose in 9 year old child
♣ There are at least four genetic variations that effect Cyp 2D6
♣ Variations cause differences in metabolism (poor intermediate extensive utra)
♣ Can either cause reduced ability to clear or activate drugs or increased activity accelerating clearance or activation
♣ 7% of Caucasians are poor metabilizers vs <1% of Asians
• Frequency of variations is different in people of different ethnic backgrounds
• Also is more common in redheads linkage disequilibrium with Cyp 2D6
o Cyp 2D6 affects metabolism of many psychiatric drugs important to test for Cyp 2D6 genetics to find the ideal dose for each individual
o NOW, dose adjustment is typically done with trial and error, in the FUTURE, genetics will probably be used to find a more ideal dose from the outset personalized medicine (family history, clinical data, genomic profile all used together)
o Ex: Tamoxifen used to treat ER+ breast cancer
♣ Cyp 2D6 metabolizes prodrug tamoxifen to MUCH more effective endoxifen
♣ Those with ineffective Cyp 2D6 activity do not effectively convert prodrug to active drug
♣ IDEA: if cancer patient isn’t experiencing any side effects, must think about genetic testing to see if patient has appropriate metabolic activity to activate drug
• Health care impact of genomics
o Pre-genomic era: disease description, uniform disease classification, patient homogeneity, universal therapy
o Post-genomic era: disease mechanisms, disease heterogeneity, individual variability, targeted therapies
• Genome testing issues
o Privacy and confidentiality, stigmatization as ‘untreatable’, need for new guidelines, incidental findings
o Personal information is not unique to the patient, it also has implications for family members
o GINA: employers, health insurance cannot discriminate based on genetic status, but life insurance, military can discriminate
o Race/ethnicity
♣ Does the emerging data of race/ethnic difference in genetic variations lead to racial profiling in health care delivery?
♣ Many issues with drugs targeted towards certain ethnic groups
• Genetic variations leading to variability in Warfarin response
o Two genes – CYP2C6 and VKORC1 – affect metabolism of Warfarin
o Mayo study found that using genetic information to determine dose led to a 30% decrease in hospitalization costs
Session 91: Cell Cycle Basics
• Normal cell replication
o Replication is mitogen- (growth factor) dependent (only divides when told to)
o Replication is anchorage-dependent (must be anchored to extracellular matrcix)
o Replication is contact-inhibited – cells normally stop growing when available space is filled
o Cells are mortal – normally have a limited number of divisions before they die (telomeres shorten)
o Cell division
♣ Complex network known an cell-cycle control system or cell cycle clock governs progression through cell cycle
• The cell cycle
o Two most basic functions
♣ DNA replication (accuracy)
♣ Chromosomal segregation (each daughter receives copy of entire genome)
o How do cells duplicate their contents?
♣ Mitochondria are very plentiful – doubling number with each cycle is sufficient to ensure nearly perfect segregation
♣ Other organelles (ER, Golgi) break into small fragments which increases their chances of equal distribution – grow in size in daughter cells
o Cell-cycle times
♣ Vary between species
♣ Examples: intestinal epithelial cells – 12 hours; fibroblasts – 20 hours; human liver cells – 1 year
o Stages
♣ M phase: mitosis, about 1 hour
♣ Interphase: about 23 hours (S-phase, chromosome duplication about 11 hours)
• Regulation
o Checkpoints:
♣ G1: Restriction Point – start checkpoint in yeast, are there growth factor signals present? Has the cell grown sufficiently?
♣ S Checkpoint – DNA damage checkpoint, DNA replication halted if genome is damaged
♣ G2/M Checkpoint – entrance into M blocked if DNA replication is not completed
♣ Spindle Assembly Checkpoint (metaphase-to-anaphase transition checkpoint) – anaphase blocked if chromatids are not properly assembled on mitotic spindle
o Cell cycle is controlled by protein kinase complexes
♣ Cyclin-dependent kinases (Ckds) – ser/thr kinases, inactive unless bound to cyclins, phosphorylates proteins involved in cell cycle when active
♣ Cyclins – have no enzymatic activity themselves, are a regulatory (activating) subunit of Ckds that direct to target proteins
• G1-cyclins (D) – induced by mitogens, regulates the activities of G1-Cdks (needed to go through restriction point)
• G1/S-cyclins (E) – help trigger progession through restriction point
• S-cyclins (A) – bind Cdks right after restriction point, stimulate chromosome duplication and control many early mitotic events
•
M-cyclins (B) – activates Cdks that stimulate entry into mitosis
♣
Regulation of Cdk Activity
• Controlled degradation of cyclin subunits, some Cdks degraded during cell cycle (each cyclin only present during specific time during cell cycle)
• Phosphorylation and dephosphorylation regulates activity
• Cdk-inhibiting proteins (CKIs, CIPs, INKs) interfere with kinase activity
• Transcription of the cyclins and CKIs
o Controlled degradation of cyclins during the cell cycle
♣ Cyclins “rule the cycle” – undergo a cycle of synthesis and degradation
♣ Cycle ensures that
• Each checkpoint is ‘checked’ at each cycle
• Only one round of cell division occurs unless mitogens still present
♣ During G1 and S phases, the SCF E3 ubiquitin ligase complex polyubiquitylate and destroys G1/S cyclins (D, E, A)
♣
In M phase, anaphase-promoting E3 ligase complex (APC/C) polyubiquitylates and destroys M-cyclins (B)
o Phosphorylation/dephosphorylation of the Cdks (ex M-Cdk)
♣ Mostly used to regulate M-Cdk activity
♣ MPF: M-Cdk in vertebrates
♣ Wee1: tyrosine kinase
♣ CAK: Cdk-activating kinase
♣ Cdc25: protein phosphotase
o CKIs Inhibit Cyclin-Cdk Activity
♣ CKIs wrap themselves around cyclin-Cdk complex to inactivate it
♣ Ex: p16Ink, p15Ink, p18Ink, p19Ink, p21Cip, p27Kip
o
Transcription of key genes regulates the cell cycle
♣ Transcription of cyclins, CDKs, etc
Session 92: Regulating the Cell Cycle
• Cyclin-Cdks required by ALL cells
o G1/S Cyclin-Cdks (cyclin E-Cdk): control entry into S phase (progression through restriction point)
♣ Phosphorylates transcription factors controlling genes whose proteins are needed for DNA replication
o S-Phase cyclin-Cdks (cyclin A-Cdk): controls DNA synthesis
♣ Phosphorylates protein components of the prereplication complexes at origins or replication
o Mitotic Cyclin-Cdks (cyclin B-Cdk): control mitosis
♣ Phosphorylates hundreds of proteins
• Fourth class required by MOST cells
o G1 cyclins (cyclin D-Cdk): control the activities of G1/S cyclins
♣ Induced by mitogens
♣ Note: mitogens not required by embryonic stem (ES) cells
• ES cells respond to an intrinsic timer or oscillator instead of mitogens
• Don’t really have a restriction point
• Only WT cells that are tumorigenic
• Cell Cycle ‘Clock’
o Integrates signals from outside and within cell to control cell cycle initiation and progression
♣ Signals include tyrosine kinase, GPCRs, TGFB, nuclear receptors, nutrient status
• G1 Checkpoint:
o M-cyclin (cyclin B) degradation and Cdk Inactivity ends M-Phase and begins G1
o Key point where cells decide whether or not to divide
o Based on whether mitogenic signals present, DNA damage, cell has grown sufficiently
o Cells enter G0 between divisions
♣ May be terminal (neurons, muscle cells)
♣ Can be quiescent state but one where cell cycle machinery is intact (liver)
♣ Can by transient, cells enter and leave rapidly (fibroblasts, intestinal cells)
♣ Imposed on all cells, at least temporarily, by degradation of the M-phase cyclin after mitosis
o Mitogens stimulate synthesis of the D-cyclins (MAPK, JAK/STAT, Wnt pathways, etc), induction of Myc, AP-1 (Fos and Jun), B-catenin, STATs, SP1…
♣
Inactivation of Rb and activation of E2F
♣ Induction of SCF and degradation of CKIs
♣ Synthesis of G1/S cyclins and other proteins needed for DNA synthesis
♣
♣ When TGFB is acting as a tumor suppressor, it opposes cell proliferation by preempting the mitogenic pathway
• Increases expression of CKIs
• Blocks phosphorylation of Rb
• Prevents expression of Myc
o Embryonic Stem ells are not subject to the G1 checkpoint
♣ Rb is usually hyperphosphorylated all the time and thus inactive
♣ Mitogens and MEK signaling not needed for progressiong G1
♣ No DNA damage checkpoint in G1
♣ Cyclin E expression is constant, not cyclical
♣ Net result: ESC pass through G1 rapidly, allowing rapid cell proliferation
• DNA damage checkpoints (G1/S-Cdk, S-Cdk, M-Cdk phases)
o DNA damage (esp single- double-stranded breaks) is detected by Kinases
♣
Activate the ATM and ATR kinases
♣ ATM and ATR activate the Chk1 and Chk2 kinases
• These phosphorylate Cdc25 phosphatase leads to degradation
• Cdc25 normally removes inactivating phosphoryl from Cdks, without this signal Cdks cannot be activated cell cycle is blocked
♣ Both ATM and Chk kinases increase p53 levels by phosphorylating p53, which kicks off inhibitor Mdm2 (remember???)
• Binds to many target genes
• Signals DNA repair
• If this fails apoptosis
• Inhibits cell cycle (CKI activation)
• Unreplicated DNA Checkpoint (M-Cdk phase)
o Also activates Chk1, inactivates Cdc25, etc
• Spindle-Assembly Checkpoint (APC/C)
o
APC/C activated degrades Securin (from Securin-separase complex) active separase cleaves cohesins that hold chromatids together
o Prolonged activation of the checkpoint cell death
o Many anti-cancer drugs target this step
Session 93: the Cell Cycle and Cancer
• One check against mitogen overstimulation: increased p53
o Excessive Myc production activates Arf (14-3-3) complexes with Mdm2 (inhibits) increased p53
• Cells can overcome their control systems
o Cancer from deregulated cell proliferation
♣ Mutations that short-circuit the need for mitogens – activating proto-oncogenes (RTKs, Cyclin D, Ras, PI3K, etc); or inactivating tumor suppressors (PTEN, p53, TGFB)
♣ Mutations that target the G1 checkpoints – inactivation tumor suppressors (Rb, CKIs, p53); mutations that increase Myc or AP-1
o Cancer from deregulated cell survival
♣ Mutations that suppress apoptosis – activating mutations in PI3K cascade, inactivating mutations in PTEN, p53
• Mutations of critical genes
o Gain of function of proto-oncogene creates ocogene – dominant only in somatic cells
o Loss of function of tumor suppressor genes – (typically, not p53) recessive in somatic and germ cells (can be heritable), can have strong tissue preference
• Common conversion of proto-oncoproteins oncoproteins
o Missence mutation in transmembrane regions of Her2/neu receptor leads to dimerization even in absence of ligand
o Deletion of external domain of EGF receptor dimerization without ligand
o Chromosome translocations can create fusion proteins with oncogenic proteins
♣ Philadelphia chromosome created by translocation of tips of 9 and 22
♣ Creates BCR-ABL fusion protein, which is a constitutively active tyrosine kinase
♣ Phosphorylates many signaling molecules, such as JAKs
♣ Leads to chronic myelogenous leukemia (CML) if occurs in bone marrow
♣ Imatinib (Gleevac) targets Abl kinase first cancer drug targeting to a signaling protein unique to cancer cells
• Mechanisms for inactivating a tumor suppressor gene
o Rb gene example (major tumor suppressor for cell cycle progression)
o All except point mutations lead to LOH
o Tumor suppressor genes can be inactivated by both epigenetic and genetic mechanism
♣
Epigenetic changes are much more common could inactivate only good copy of gene
•
Why isn’t cancer more frequent?
o 1016 cell divisions occur in humans in a lifetime
o Spontaneous mutations in a carcinogen-free environment occur about 10-6bp/gene/cell div
o In a lifespan, each gene is likely to have been mutation 1010 times
o Multiple genetic events are needed
o Cancers are though to arise from a single cell with more than one mutation
• Evidence supporting Multi-Hit Model of cancer induction
o All of the cells in a tumor should have at least some genetic alterations in common
♣ Supported by microarray analysis; in female tumors all cells have same X-inactivation
o Cancer incidence increases with age more chance for multiple mutations to occur
o In mouse models, even with overexpression of potent oncoproteins, cancer initiation is extremely slow unless more than one is introduced
o Successive mutations have been traced in colorectal cancers
• Mutations in p53 are particularly devastating – ‘guardian of the genome’
o Reponds to many different signals (lack of nucleotides, UV damage, hypoxia, etc)
o Promotes cell cycle arrest when DNA is damaged
o Triggers DNA repair mechanisms
o Initiates apoptosis when damage is too severe
o Blocks angiogenesis, excessive mitogen signaling
o p53 functions as a tetramer, mutations in only one allele of TP53 can cause cancer
♣ Mutations that inactivate p53 usually occur in DBD typically recessive, usually require both alleles to be mutated, but in some cases can bind to WT p53 in dominant fashion
♣ Other mutations are in the oligomerization domain dominant negative mutations, only one allele needs to be affected
♣ Ex: Li-Fraumeni syndrome – individuals with this disease develop tumors early in life (AD)
♣ 50% of cancers have mutations in p53, other 50% have mutations in p53 regulators (Chk1)
• Characteristics of Cancer Cells
o Abnormally high mitotic rate - used to estimate malignant potential
o Signs of de-differentiation and assume features of immature stem cells
o Disordered growth – not subject to contact inhibition and grown in a sprawling mess
♣ Anaplasia – de-differentiation and disordered growth, degree of anaplasia is predictive of malignant behavior, patient’s survival
o Can metastasize – ability to migrate, cross basement membrane, grow in a strange environment
o Are genetically unstable – keep mutating because they cannot prevent or repair DNA damage
o Are immortal
o Become self-sufficient for growth and proliferation
o Induce help from local stromal cells
o Induce angiogenesis
Session 95: Cancer Genetics
• Cancer Basics
o Common – 1/3 of population will have cancer in lifetime, 20% of deaths in developed nations
o Early diagnosis improves outcome
o Cancer causes (sporadic most common)
♣ Sporadic – frequently only one person in family with cancer, no germline mutation
• Typically occur later in life
♣ Familial – number of primary/secondary relatives with cancer, no evidence of a cancer syndrome or germline mutation
♣ Hereditary – heritable germline mutation, inherited in autosomal dominant manner
o Multistep process, requires accumulated sequential mutations
• Oncogene
o Mutations in genes that normally function to promote cell survival or limit cell death
o Examples: telomerase, Bcl2, Myc
o Typically cause cancer by a gain of function mutation
• Tumor Supressor Genes
o Gatekeepers – control cell growth by regulating cell cycle checkpoints or promoting apoptosis
o Caretakers – guardians of the cell’s genome, correct normal day to day errors that occur in genome
o Typically cause cancer by loss of function mutations
• Hereditary Cancers
o Autosomal Dominant
o Penetrance is not 100% but quite high
o If the mutation is in a tumor suppressor gene, requires a second hit for the cancer to start
o Frequently cell type (cancer type) specific but may have patterns of tumors that would be indicative of the cancer syndrome
o Tend to be bilateral in paired organs (breast cancer, retinoblastoma)
o Tend to be early onset
o Hereditary 54 described hereditary cancer syndromes
• Retinoblastoma
o Caused by germline mutations in the Rb gene, requires second Rb mutation to develop tumor
♣ Rb encodes a gatekeeper
♣ Inherited in dominant manner, but in the cell it is recessive (as second mutation must happen)
o Clinical presentation
♣ Very early onset: sometimes present at birth
♣ Tumors can be bilateral and at multiple sites in retina
♣ Later in life, individuals are at risk for osteosarcoma
• Colon Cancer Syndromes
o Familial Adenomatous Polyposis (FAP)
♣ Autosomal dominant mutations in APC gene controls action of beta-catenin in Wnt pathway
♣ Diagnostic charactertics
• >100 colorectal adenomatous polyps (typically in 1000s)
• <100 and a relative with FAP
• Attenuated (milder) forms may have fewer polyps
• Onset of polyps early in life; median age is 16 years (range 7-36 years)
♣ Polyps are first benign progress to cancer
♣ >95% with APC will develop cancer
♣ 12% will develop duodenal carcinoma
♣ Screening (sigmoidoscopy), many individuals eventually have colon removed
o Lynch Sydrome (Hereditary Non-polyposis Colon Cancer, HNPCC)
♣ Early appearance of colon cancer (average is 43, sporadic is typically early 60s)
♣ Family history of colon/endometrial cancer
♣ Cancers grow quickly and are aggressive
♣ Mutations in four genes: MLH1, MSH2, PMS2, MSH6 (possibly MSH3, PMS1)
♣ Genes involved in mismatch repair
• Hallmark ‘microsatellite instability’ – DNA slippage events in short repeat units are not repaired, show slightly different microsatellites sizes when run on gel
♣ Cancers in lynch syndrome
• Colon cancer: tends to be ‘right-sided’, risk of 70% by age 70
• Endometrial cancer: 30-60% by age 40
• Other tumors with a high percentage: gastric, biliary, ovarian, urinary tract, etc…
• Requires screening with consideration of removal of colon, uterus, ovaries
• Li-Fraumeni Syndrome
o Caused by mutations in p53 (TP53)
♣ TP53 causes cell cycle arrest that is required to permit repair of damaged DNA
o Clinical presentation
♣ Early onset cancer syndrome with multiple primary cancers throughout lifetime
♣ 90% incidence of cancer by age 70
o Cancers
♣ Breast cancer 25%
♣ Rhabdomyosarcoma 20%
♣ Brain cancer 13%
♣ Adrenocortical carcinoma 10%
♣ Osteogenic, chondrosarcoma 15%
• Hereditary Breast Cancer Syndromes
o BRCA1 and BRCA2 mutations found in 2/3 of families with 3+ individuals with breast/ovarian cancer, found in 10% of women regardless of family history
o High frequency of BRCA1/2 founder mutations in Ashkenazi Jewish women
o Lifetime risk of breast cancer (men are also at risk for breast cancer)
♣ BRCA1 by age 70
• 65% will have breast cancer, 39% will have ovarian cancer
♣ BRCA2 by age 70
• 49% will have breast cancer, 18% will have ovarian cancer
o Role of BRCA1/2
♣ BRCA1 – multifunction E3 ubiquitin ligase involved in DNA damage, signaling, DNA repair by homologous recombination
♣ BRCA2 – repair of double stranded breaks through homologous recombination
o Cancer screening
♣ Monthly self examinations starting at age 18
♣ Clinical examination twice yearly starting at age 25
♣ MRI and mammogram yearly starting at age 25
♣ Twice yearly transvaginal ultrasound/CA125 blood test (ovarian cancer marker)
♣ Consideration of salpingo-oophorectomy after age 35-40 or childbearing
♣ Consideration of prophylactic bilateral mastectomy
Session 97: Ethics and Genetic Counseling
• Ethics
o Establishment of a set of guidelines for morally acceptable conduct within a theoretical framework
o Principles
♣ Beneficence – benefit
♣ Nonmaleficence – no harm, for example doing research/clinical trials
♣ Autonomy – rule of self, patient makes independent decisions
♣ Justice – equal care
• Ethical/professional issues in genetics
o Informed consent – to make a decision, patient needs to understand treatment, other options, privacy issues, etc…
♣ Examples: predictive testing for adult onset diseases (ALS, BRCA), testing minors, research
o Confidentiality – situations in which a professional must decide whether or not to share privileged information with family members, insurance companies, fellow professionals, third parties
♣ Example: duty to warm family members of risk, must counsel patient about this
o Withholding information – situations in which a professional must decide whether or not to share information with patient
♣ Examples: unanticipated information (non-paternity, whole genome sequencing), withholding is in patient’s best interest?
o Uncertainty – situations where there is ambiguity about how genetic information
♣ Examples: test results from cancer susceptibility test (lack of data), variants of uncertain significance
o Value Conflicts - situations where there is a difference in a personal/professional values
♣ Examples: sex selection, patients selecting for such traits as deafness of dwarfism
o Directiveness – to what extent should a professional influence a patient’s decision making
o Discrimination – situations in which a professional or patient is concerned about unfair treatment by an insurance company or employer based on results of a genetic test
♣ MN statute and GINA protect in some circumstances
o Diversity – situations in which ethnicity, religion, socioeconomic status, language etc present an obstacle in the genetic counseling process
o Who is the patient? – situations in which parents disagree about what to do with respect to child or families disagree about procedures/testing
o Documentation – illegal not to record, even if patients doesn’t want it to be
Session 99: Gametogenesis and Fertilization
• Sexual Reproduction
o Advantages: Genetic variation and elimination of deleterious genes (through sexual selection)
o Timing, onset, duration of meiosis are sexually dimorphic
• Retinoic Acid (vitamin A derivative) regulates the timing of entry into meiosis
o RA binds to RAR, a nuclear receptor
o Females: in embryonic ovary, high levels or RA induce proliferating germ-line cells to enter meiosis at about 11-12 weeks of gestation
o Males: in embryonic testis, sertoli cells produce an enzyme (Cyp26b1) that metabolizes RA, only after puberty do enzyme levels decrease, allowing RA levels to rise and meiosis to resume
• Gametes specialization
o Sperm: very small, highly motile, no organelles except for mitochondria, highly competitive
o Ovum: very large, nonmotile, contains many materials/organelles to support embryo growth
•
Spermatogenesis
o Sperm develop as a syncytium – connected together to synchronize development and so X containing sperm get same proteins as Y containing sperm
o Acrosomal vesicle contains hydrolyic enzymes to penetrate ovum’s outer coat
o DNA is tightly condensed by protamines and sperm-specific histones
o Sertoli cells (nurse cells)
♣ Secrete anti-Mullerian hormone (AMH)
♣ Convert testosterone to estradiol
♣ Form blood-testes barrier
♣ Secrete inhibin and activins after puberty to regulate FSH secretion
o Leydig Cells (interstitial cells)
♣ Produce testosterone in response to LH
o Sperm are not capable of fertilization until they undergo capacitation in the female genital tract
♣ Occurs in fallopian tubes, takes 5-6 hours
♣ Involves extensive biochemical and functional changes in the sperm
• Cholesterol efflux, membrane becomes hyperpolarized, increased cytosolic pH, unmasking of cell surface receptors that bind the sperm to the ovum
♣ Stimulus for capacitation is unclear, in vitro fertilization requires albumin, Ca2+, HCO3-
• Albumin helps extract cholesterol, increasing the ability of plasma membrane to fuse with acrosome during acrosome reaction
• Ca2+, HCO3-Eenter the sperm and activate a soluble adenylyl cyclase enzyme to increase cAMP leads to tyrosine phosphorylation of many proteins
•
• Ovum maturation
o Oocyte: developing egg/ovum that cannot bind sperm of be fertilized
o Egg = mature ovum = ovum: capable of being fertilized, arrested in metaphase II
o The primary oocyte uses special strategies to grow:
♣ Most of the growth occurs when the cells are bivalent (4N), so there is twice as much DNA to use for RNA synthesis
♣ Some genes such as ribosomal RNA genes become amplified
♣ Follicle cells link to oocyte via gap junctions to provide precursors for protein synthesis
o Ruptured follicle corpus luteum secretes progesterone to maintain pregnancy
o
Gonadotropins FSH and LH regulate Ovarian and Testicular Development
♣ During reproductive years, LH and FSH stimulate ovulation
♣ During puberty, Kisspeptin is the key regulatory of the onset of puberty
• It binds to a GPCR in the pituitary (GPR54)
• This signals the pituitary to release LH and FSH
• FSH drives maturation of Sertoli, Granulosa cells
• LH stimulates Leydig, thecal cells
o Regulation of Meiosis in Mammilian Oocytes
♣ Meiotic arrest depends on high cAMP levels in oocyte
♣ The FSH/LH surge acts on granulosa cells to trigger the oocyte to resume meiosis
♣ CNP and NPR-B (natrieretic peptide receptor) contribute to maintaining meiotic arrest
•
NPR2 GTP to cGMP inhibits PDE3A stops degradation of 5’-AMP
• Fertilization
o Around 300 million sperm are deposited at the opening to the cervix
o Only about 200 reach the ovum
o Ovum surrounded by glycoprotein matrix called zona pellucida and layer of follicular cells called corona radiata
o Meeting of sperm and ovum
♣ Ovum secretes chemoattractant peptides
♣ Sperm binds to the zona pellucida
♣ Sperm passes through ZP, contains glycoproteins ZP1, ZP2, ZP3 (may be sperm receptor)
♣ ZP induces the acrosomal reaction, contents of the acrosome are exocytosed in a Ca2+ mediated reaction
o Blocking Polyspermy
♣ Fast reaction: electrical potential of the ovum membrane is changed within 1-3 seconds as Na2+ enters ovum lasts 1 min (not detected in mammals)
♣ Slow reaction: the cortical granules fuse with the ovum membrane, modify proteins in the ZP including ‘sperm receptor’ ZP3 cortical reaction
o Ovum activation after sperm binding begins development program (involves PLC pathway Ca2+ ^^)
♣ Exocytosis of cortical granules
♣ Resumption of meiosis and extrusion of 2nd polar body
♣ Release of inhibition on maternal mRNAs
♣ Stimulation of protein synthesis, DNA replication, etc
o Sperm contributes centrioles needed for first mitotic division
Session 100: Sex Determination and Differentiation
• Primordial Germ Cells (PGCs)
o PGCs are the cells that give rise to the gametes
o PGC specification begins at gastrulation, initiated by high levels of BMP family members (in mouse)
♣ Part of TGFB family, bind to Ser/Thr kinase receptors
o Specification: the first stage of commitment of a cell or tissue to its fate
• Mammalian Gonad
o Bipotential (indifferent) – can become male or female from weeks 4-7
o Sex chromosomal composition of the somatic cells surrounding the PGCs determines differentiation
o Male: wolffian duct, XY or XXXXXXXXXXXY
o Female: Mullerian duct, XX or X0
• Development of Male Phenotype
o Testis determination controlled by SRY (sex determining region of the Y chromosome) on short arm of Y chromosome
♣ Encodes 223 aa tranction factor, expressed in a number of tissues in fetal development
♣ Activates the male-specific transcription factor Sox9
♣ Somatic cells expressing Sry gene become sertoli cells
o Sertoli Cells direct development
♣ Stimulate PGCs to develop along pathway that produces sperm and inhibit from entering meiosis, which oocytes must do
♣ Secrete AMH (anti-Mullerian hormone) causes regression of Mullerian duct
♣ Stimulate development of other somatic cells to become Leydig cells
♣ Leydig cells secrete testosterone, induces male reproduction structures and secondary sexual characterstics and masculinizes the brain
o
Inititation of sex determination
♣ In the default female pathway, paracrine signals and transcription factors activate Wnt4 and R-spondin1
• Wnt4 increases beta-catenin, which inhibits Sox9 male pathway cannot proceed
♣ If Sry is present, it acts with Sf1 to induce Sox9
• Sox9 induces Fgf9, stimulates testis development and feeds back to increase Sox9
o Fgf9 – growth factor, binds to tyrosine kinase receptor
• Sox9 inhibits beta-catenin’s activation of ovary
o Mechanism of action of Sry
♣ Contains two nuclear locatization domains, must be bound by CaM and Impbeta to move into nucleus
♣ Sry and Sf1 bind directly to enhancer region (TESCO) of Sox9 gene induce expression of Sox9
♣ Sox9 also binds directly to TESCO site with Sf1 to amplify Sox9 expression
o Sox9: the autosomal testis-determining gene
♣ Transcription factor that induces testis formation in all vertebrates
♣ Functions
• Upregulates its own promoter, prolonging its own expression
• Blocks ability of beta-catenin to induce ovary formation
• Regulates numerous genes requires for testis formation – AMH, FGF9
♣ NOTE: although Sry is the trigger for male sex determination, Sox9 orchestrates and stabilizes Sertoli cell differentation to lock in the testis-determining program
o FGF9 is also essential – induces proliferation of Sertoli cell precursors and stimulates their differentiation, without sertoli cells, the male structures do not form
o Sf1 (steroidogenic Factor 1) – link between Sry and male development pathways
♣
Nuclear orphan receptor (ligand not known)
♣ Binds to DNA in monomeric form
♣ Activates AMH in sertoli cells and the enzymes that make testosterone in Leydig cells
♣ Individuals heterozygous for Sf1 have malformed gonads, retain Mullerian duct
• Development of Female Phenotype
o Initiation of sex determination (see figure above)
♣ Female pathway is default, Wnt4, Rspo1 (inhibits Wnt inhibitor)beta-catenin inhibits Sox9
• Beta-catenin prevents Sf1 from binding to TESCO enhancer
♣
Wnt4, R-spondin1 required for normal ovarian development
•
Sex determination vs. differentiation: development of female and male phenotypes in response to hormones secreted by the gonads
Session 101: Disorders of Sex Development
• Disorders of sex development
o Genotypic sex based on presence of the type of sex chromosomes and the phenotypic sex are disconcordant or external genitalia are sufficiently ambiguous to preclude sex assignment at birth
o Umbrella term that supplants terms like intersex, hermaphrodite, etc…
o Many are characterized by ambiguous genitalia: hypospadias, chordee, absent testes, enlarged clitoris, masculinized labia majora, extrophic bladder
• Terms
o Wolffian structures: male structures – epididymis, vas deferens, seminal vesicles
o Mullerian structures: female structures – fallopian tubes, uterus, upper 2/3 vagina
• Sex Chromosome Disorders
o
Turner Syndrome
♣ 45, X (most common); 46, X, iso Xp (has two short arms); 46, x, iso Xq; X translocation to an autosome or Y chromosome, 45, X/46, XY mosaicism (ratio determines sex characteristics)
♣ 1/5000 live births
♣ Clinical characteristics:
• Short stature, infertility, congenital heart disease, streak gonads (no follicle cells in ovaries), kidney malformations, hand/food edema, neck webbing, wide carrying angle, low hair line, wide spaced nipples, late onset hearing loss
o Klinefelter Syndrome
♣ 1/500 males
♣ 47, XXY
♣ Clinical characteristics
• Tall stature with long limbs, small testes, low testosterone – leads to under-virilization and decreased bone density, azoospermia, learning disability or behavioral changes
o 46, XX/46, XY Chimerism
♣ Two cell lines that occurred from different zygotes
♣ Phenotype depends on percent and location of one cell type vs another
• 46, XY Disorders
o Partial and Mixed Gonadal Dysgenesis (PGD)
♣ Characteristics
• Ambiguous genitalia, mild to severe penile scrotal hypospadias, dysgenetic testes may become tumors later in life, reduced to absent sperm production, various degrees of both Mullerian and Wolffian structures
o Complete Gonadal Dystenesis (CGD)
♣ Individuals appear as normal woman and may not present until puberty when they fail to develop menses
♣ Completely underdeveloped gonads, need to be removed by adulthood to prevent tumors
♣ Normal Mullerian structures
♣ Woman are infertile, but may carry a donated pregnancy
o Genetic basis
♣ Deletions of small regions on 9p, 2q, 10q
♣ Duplications of 1p (Wnt4) or Xp (Dax1 aka NROB1)
♣ Single gene mutations
• Sry – 1% of 46, XY PGD; 15% of 46, XY CGD
• Sf1 – 13% of 46, XY DSD
• DHH – 20% DSD, 50% CGD
• WT1
• 46, XX Disorders
o Testicular Disorder of Sex Development
♣ 46, XX Karyotype
♣ clinical findings
• male appearance (80%) or ambigiuous male (20%), two testicles, no Mullerian structures, low testosterone, gynecomastia
♣ Genetic basis
• Translocation of Sry from Y to X chromosome found in majority
• De novo event
o Ovotesticular Disorder
♣ Formerly known as True Hermaphroditism
♣ Causes are variable: chimerism – 46, XX/46, XY; mosaicism, etc
♣ Characteristics
• Mullerian and Wolffian structures may both be present
• Hormonal basis of DSD
o Abnormalities of sex differentiation
♣ In production or response to hormones that are produced by the testes, adrenal glands, or other sources that change the external appearance of genitalia
o Congenital adrenal hyperplasia (CAH)
♣ Mutation in genes that encode enzymes needed for the biosynthesis of cortisol
♣ Most common is 21-hydroxylase deficiency
♣ Have increased 17-hydroxyprogesterone (used to screen)
♣ Characterstics: virilized female genitalia – ambiguous genitalia appears male
♣ Infants can have salt wasting – leads to death in first days of life if not treated
♣ Common – 1/10000 in Europe, 1/300 in Yupik Eskimos
o 5 alpha-reductase deficiency
♣ 46, XY
♣ Ambiguous genitalia, testes present in abdomen or inguinal canal or labia, virilization at puberty due to effect of testosterone
♣ Most individuals are raised as girls but adopt male identity after puberty
♣ Testosterone DHT by 5-alpha reductase, induces transcription of proteins needed for male differentiation
o Complete Androgen Resistance
♣ 46, XY but unambiguous female
♣ Mutation in gene encoding the Androgen Receptor in Xq11-12
♣ Testosterone is produced but cells cannot respond to its signal
♣ 2-5/100000 individuals (fairly rare)
♣ Individuals have normal breast development at puberty, testes present in abdomen or inguinal canal or labia
♣ Most individuals raised as women
• Sex assignment
o 2/10000 babies are born with ambiguous genitalia
o Both a medical and a social crisis
o Do not risk assignment until you have all the data you need to help the child
o Testing needed:
♣ Karyotype, steroid measurements, electrolytes to rule out salt wasting, blood pressure, ultrasound or MRI of pelvis
o Final assignment may take days, and genotypic sex may not be the same as phenotypic sex
• Surgical Management
o Controversial intervention
o Arguments against early surgery
♣ Young girls have no use for ‘functional’ vagina until menses, intercourse
♣ We could reduce total number of operations needed to achieve vaginal length, while reducing risk of stenosis and give patients greater control over their lives
♣ No evidence that early surgery improves gender or psychosocial development
o Arguments for early surgery
♣ There is more mobility of infant tissue
♣ There is a shorter pelvis length that allows easier mobilization of urogenital sinus
♣ Many still argue that psychosocial rearing/bonding with parents is easier (no evidence)
o Consensus
♣ Surgery recommended for patients with high confluence by 2-6 months of age
♣ Never remove clitoris, preserve neurovascular bundles
♣ Females with CAH have low risk for gender identity problems
o Evaluation before surgery
♣ Defining vaginal confluence with UG sinus is most important step
♣ Genitography is recommended for pre-operative planning
o Procedure
♣ Goals are to recreate normal appearance and function external genitalia
♣ Preserving bladder function
Session 102: Genetic Association Studies
• High sensitivity = genotype catches all cases, Low Specificity = many unaffected have same genotype
• Low sensitivity = miss the correct genotype for cases, High specificity = avoid misdiagnosing unaffected
• Clinical utility
o Is it sensitive and specific?
o Does it provide predictions of disease occurrence and outcomes?
o Does it replace or contribute to other clinical diagnostic tests?
o Is it cost effective?
• What tools do we have?
o Genome variations associated with disease (DNA)
♣ Genome Wide Association Studies – GWAS
• Manhattan plot shows SNP association with disease vs control
• Pros: high throughput screening with no needed hypothesis, use of haplotype blocks and LD can simplify search, can show significant association even if low penetrance
• Cons: multiple testing error = 1/20 association may occur by chance, need to overcome false positives by large case-control numbers, may identify a haplotype block not a gene or specific cause, OR may be low association, may have significant association and NO UTILITY
♣ Make use of HapMap information
o Gene expression profiling (mRNA)
o Whole genome sequencing
Session 103: Cancer Cytogenetics
• Constitutional Abnormalities – germline; in most cases, arise during meiosis
• Acquired Abnormalities – acquired in association with the development of a malignant process (sometimes in utero, usually after birth)
• Most cancer cells have associated chromosomal abnormalities
o Abnormalities are acquired, clonal (two or more cells have the same abnormality), limited to the tissues involved in the malignancy
o Identification of abnormality is important for diagnosis, (gene-product targeted) therapy, prognosis
• t(9;22) translocation
o Example karyotype: t(9;22)(q34;q11.2)
o Diagnosis: Chronic myelogenous (myeloid) leukemia (CML)
o Derivative chromosome 22 is called Philadelphia chromosome Ph+
o Translocation results in fusion of the breakpoint cluster region (BCR) in 22q11.2 to the Abelson murine leukemia virus oncogene (ABL1) in 9q34
♣ BCR-ABL1 fusion on 22 is Philadelphia chromosome
♣ ABL1-BCR on 9 is not involved in leukemia
♣ Can be detected by FISH
♣ Translocation results in the formation of a novel (chimeric) BCR-ABL gene that encodes a protein with altered tyrosine kinase activity
o Cytogenetics used in CML
♣ Confrism diagnosis, determine stage, monitor response to therapy
o Natural history of CML
♣ Chronic phase: t(9;22) is typically the sole abnormality, symptoms very mild
♣ Accelerated phase: 75% of cases gain additional abnormalities (e.g. +8, i(17)(q10), +der(22)t(9;22)), secondary abnormalities are no consistent but some are commonly seen
♣ Blast crisis: additional abnormalities typically present as in accelerated phase, lethal if not treated
o Current therapies
♣ Hematopoetic stem cell transplant
♣ Gleevec - Therapy targeted at the specific abnormal tyrosine kinase generated by the t(9;22)
• Don’t know if individuals need to take therapy their whole lives
• t(15;17) translocation
o Example karyotype: t(15;17)(q24.1q21)
♣ PML (promyelocytic leukemia) gene on 15q24.1 fuses with RARA (retinoic acid receptor alpha) on 17q21
♣ RARA transcription factor regulating transcription of genes important in the maturation of white blood cells beyond the promyelocyte stage
♣ PML acts as a tumor supressor
o Diagnosis: Acute Promyelocytic Leukemia
♣ Arrest in development of promyelocytes, have fibrous-looking rods in cytoplasm
o Recognition of the PML-RARA fusion is critical for care
♣ APL associated with coagulopathy
♣ Targeted therapy: ATRA + Arsenic + chemo
♣ Recent study in blood: 98% sustain complete remission
♣ ATRA should be given within 24 hours of diagnosis – given to patients when PML is suspected
• t(4;11)(q21;q23) translocation
o Presentation: somewhat elevated WBCs, very high circulating blasts, anemia
o A specific recurring abnormality
♣ Accounts for 60% of cases of acute lymphoblastic leukemia of infancy
♣ Very poor prognosis – immediate planning for bone marrow transplant
o MLL gene is a human homolog of Drosophila trithorax gene that regulates HOX genes
o AF4 is thought to be involved in lymphocyte development
• High hyperdiploidy (>52 chromosomes)
o Associated with B-cell lineage acute lymphoblastic leukemia of childhood
o Very favorable prognosis
o Presence of trisomies for 4,10 now used to stratify to a ‘low risk’ leukemia therapy group
• Case 5: 22 yr old female, B-acute lymphoblastic leukemia
o Presentation: hypercellular marrow, very high blasts, B markers present, normal G-banding cytogenetics, normal FISH for the recurring abnormalities
o Array showed 21 abnormalities not shown through G-banding
o Found 7 important abnormalities
♣ Small deletion in 7p12.2: IKZF1 gene
• Associated with increased risk of relapse and adverse events
• 74% will relapse
♣ Small deletion in 9p13.2: PAX5 gene
• Recent studies show some patients with high-risk, active lymphoblastic leukemia have mutations affecting tyrosine kinase and cytokine signaling can be targeted
• HER-2/neu
o c-erb B2
o A proto-oncogene
o Encodes a tyrosine kinase receptor
o The ligands of HER-2/neu and related growth factor receptors are known as heregulins
o HER-2/neu amplification
♣ Pts with multiple copies of the gene in tumor tissue had a shorter time to relapse and a shorter overall survival
♣ Amplification occurs in 25-30% of human breast cancers, also in some ovarian malignancies
o Therapy
♣ Use of recombinant anti-HER-2 monoclonal Ab (Herceptin/trastuzumab) together with cisplatin clinical response in patients with HER-2 overexpressing metastatic breast cancer refractory to other chemotheraputic regiments
♣ mAb therapy may also increase efficacy of radiotherapy and other chemotherapeutic agents
Session 104: Stem Cells
• Two major categories of cells in adults
o Cells that are formed to last a lifetime (ex. Auditory hair cells)
o Cells that are replaced
♣ By simple duplication: differentiated cells divide (ex. Differentiated hepatocytes in liver, beta cells in pancreas)
♣ From stem cells: to replace cells that undergo rapid turnover (ex. Blood, skin, intestine)
• Stem cells
o Cells that can reproduce themselves as well as generate specific types of more specialized cells
o Properties
♣ Can undergo endless asymmetric cell division
♣ No replicative senescence (telomerase continually expressed)
♣ Each daughter cell can remain a stem cell or commit to a pathway that leads to terminal differentiation
♣ Self-renewal is the ability for a cell to proliferate in the same state
♣ Note: Rb is always phorphorylated (remember??)
o Asymmetric division
♣ Not fully understood
♣ Can be environmental (due to different environment of daughter cells) or divisional (division of RNA, proteins is asymmetric between daughter cells)
• Progenitor cells (transit amplifying cells)
o Staged between stem cels and differentiated cells
o Also called transit amplifying cells since they usually divide while ‘transiting away’ from the stem cell niche
o Related to stem cells but do not have the unlimited capacity for self renewal
o Usually more differentiated than stem cells and have become committed to a particular cell type
o Use of progenitor cells keeps the number of stem cells low and slowly dividing
♣ Reduces the potential for genetic damage and cancer
o Example: Hematopoietic Stem Cells
• Stem cell ‘Potency’
o Totipotent: can form every cell type including the trophoblast cells of the placenta (zygote)
o Pluripotent: can form every cell type except trophoblasts (ESCs)
o Multipotent: can form a limited number of adult cell types
o Unipotent: can form only one cell type
• Normal stem cells grouped into:
o Embryonic (pluripotent) stem cells: capable of developing into all cell types of the body, isolated from inner cell mass of blastocysts
o ‘adult’ stem cells: involved in replacing and repairing tissues of a particular organ, can typically forms only a limited subset of cell types, multipotent or unipotent
• Adult Stem cells
o Many/most? Adult organs contains committed stem cells
o Difficult to identify, isolate, purify as they are rare (estimated .1-3% of cells)
o
Low rate of cell division, do not proliferate readily
o In use medically ex: bone marrow transplants are essentially stem cell transplants
o Tissues and organs that undergo continual renewal (skin, intestine, breast, blood, etc) contain stem cells in areas known as adult stem cell niches
♣ Allows for controlled stem cell proliferation, differentiation of progeny that leave niche
♣ Produce paracrine factors that regulate proliferation, prevent differentiation, when cells leave niche they begin differentiation
♣ Intestinal niche: Wnt signaling maintains stem cell niche
• Other sources of multipotent stem cells:
o Mesenchymal Stem cells (MSCs)
♣ Multipotent
♣ Found in several adult tissues: bone marrow, adipose, dental pulp, breast milk, intestine
♣ Able to give rise to numerous mesenchymal (stromal) cell types: bone, cartilage, muscle, fat
o Amniotic epithelial cells: from the amniotic membrane in human term placenta
♣ Do not express telomerase and therefore do not make teratomas after transplantation
♣ Express markers that are present on ESCs, can differentiate into 3 germ layer in vitro
♣ Not used extensively now but might prove to be a source of SCs for tissue regeneration
o Fetal stem cells: found in the organ of fetuses
♣ Are somewhat more differentiated than ESCs, generate a limited number of cell types
♣ Do not form teratomas in vitro
♣ Up to 12 weeks, cells have less chance of rejection than cells derived from umbilical cord, marrow
♣ Even more controversial than ESCs
o Umbilical cord stem cells: Derived from the umbilical cord epithelium and cord blood
♣ More primivitive subpopulation of mesenchymal stem cells than bone marrow, less likely to generate host response
• Embryonic stem cells
o All cell types can be generated (in theory) from ES cells
o Most differentiated cells express only 10-20% of genes, ESCs express 30-60%
o Thought to be due to accessible chromatin structure
o Low-level expression of many cell surface receptors, enabling them to respond to many signals
o DNA methylation pattern is critical
♣ DMRT1 remethylates DNA after it becomes demethylated in the zygote
♣ Remethylation is required for pluripotency
o ES cells rely on ‘master’ transcription factors
♣ Oct4, Sox2, Nanog activate genes encoding proteins and miRNAs for self-renewal and pluripotency and repress genes that induce specific differentiation pathways
• Possible use of ES cells therapeutically to restore or replace damage tissue research and controversy
o Undifferentiated ES cells can form teratomas (can contain hair, teeth, bone, eyes, limbs, etc), so ES cells must ALL be differentiation before implantation
o Primary experimental sources
♣ Therapeutic cloning (somatic cell nuclear transfer = SCNT)
• Involves replacing the genome of an oocyte with that of an adult cell
• Need an oocyte donor and a nuclear donor
• Remove egg remove spindle apparatus transfer nucleus into enucleated egg egg and cell fused with electric current culture embryo
• Ex: Dolly the sheep, BUT she died, thought that she was actually the chronologic age of the mother because of telomere shortening
• Advantages: reduces ethical concerns as doesn’t involve ESCs, no need to identify and clone genes to screen for traits of interest
• Disadvantages: very inefficient, clones have medical problems (bad epigenetic programming), human oocytes manipulated to SCNT don’t develop to blastocyst stage, some concerns about obtaining human oocytes
o One study was able to get to blastocyst stage by injecting fibroblast nucleus into haploid cell triploid cell
♣ Excess eggs from in vitro fertilization (IVF)
• Ethical debate regarding the use of ESCs from IVF embryos
• Pro: ESC research fulfills ethical obligation to alleviate human suffering, IVF embryos will be discarded anyway
• Against: ESCs are taken from a blastocyst that is then discarded (murder?), risk of commercial exploitation of participants, ESC research will lead to human cloning
Session 105: Stem Cells and Disease
• Current role of stem cell therapy in regenerative medicine
o Bone marrow, umbilical cord, and peripheral blood stem cells are the only SC therapies routinely available
♣ Bone marrow transplant has been used to treat leukemias, other blood disease for 30 years
• Recently being used experimentally for other diseases: epidermolysis bullosa
♣ Umbilical cord has a relatively high number of MSCs, less prone to rejection
♣ Peripheral blood SCs can be used instead of bone marrow, less invasive to obtain
• Potential role of stem cell therapy
o Repair of degenerating or lost tissues
♣ Repair of neurons for spinal injuries, cardiac muscles after MI, neurodegenerative diseases
o Gene therapy for diseases
♣ Current treatment for muscular dystrophy, Type 1 diabetes, leukemias and other hematopoietic disorders, but potential for treatment of many other diseases
• The ‘Selling’ of Stem Cells
o Illegal since 1984 to sell any body parts (National Organ Transplant Act)
♣ BUT does not apply to blood stem cells obtained by apheresis – used in 2/3 BMTs
♣ Problem for poor because of pressure to sell body parts (blood, plasma not covered)
o Why allow the sale of stem cells (or body parts)?
♣ Very difficult to obtain matches, especially for mixed race individuals
♣ Children with with leukemia and aplastic anemia are in desperate need of stem cells
• Induced pluropotent stem cells (iPCSs) – most prominent type of stem cell therapy
o
Can be ‘made’ from multipotent cells by forcing the expression of certain transcription factors
o First done in 2006 by Shinya Yamanaka (nobel prize in 2012) with cocktail of 4 transcription factors
♣ Transformed a mouse fibroblast into a cell that appeared identical to an ES cell
♣ Transcription factors c-Myc, Klf4, Oct-3/4, Sox2
• Klf4 binds to beta-catenin, activates telomerase gene
• Oct-3/4 induce Sox2, which induces Nanog and Fbx15
o Note: dedifferentiation and regeneration has been known for some time in lower organisms
♣ Ex: ‘immortal jellyfish’ dedifferentiates back into an amoeba-like blob and then regenerates back into a jellyfish
o Testing for pluripotency
♣ When aggregated together, cells form a teratoma: tumor-like structure with all 3 germ layers
♣ Transcription and DNA methylation pattern were found to be almost identical to that of normal mouse ESCs (the concordance between the two varies with the cells and the labs)
o Curing a human disease in mouse using ePSCs
♣ Humanized sickle cell anemia mouse model
• Harvest tail tip fibroblasts infect with Oct4, Sox2, Klf4, c-Myc viruses correct sickle-cell mutation in iPS cells by specific gene targeting differentiate into embryoid bodies transplant corrected hematopoetic precursors back into irradiated mice
♣ In vivo reprogramming of adult pancreatic exocrine cells to beta-cells
• NGN3, Pdx1, Mafa transcription factors put into viral vector
• Injected vector into exocrine pancreas cells ‘exocrine’ pancrease cells produce insulin like beta-cells and corrected diabetic phenotype!
o Technical considerations for making iPSCs
♣ Choice of factors, methods of factor delivery, choice of cell type, parameters of factor expression, derivation of conditions, identifications of iPSC colonies, expansion and characterization
♣ Only about 1% of the cells de-differentiate completely, making it rather inefficient
♣ Regulation of expression is key – difficult to regulate post-translationally regulated events
o Uses of iPSCs – huge potential in medicine!
♣ Cell/organ therapies
♣ Disease modeling – neurological diseases, cardiovascular modeling, hepatic
• Can use diseased iPSCs to study disease (better than mouse models)
♣ Drug development – especially for mixed races and races not commonly tested
♣ Regenerative medicine
• Can generate early stem cells that have the exact genotype of the patient
• Can theoretically correct genetic diseases
• Minimal ethical issues
• Propensity to be tumorigenic (readily forms teratomas as with ESCs)
• Likely to be more tumorigenic because of use of viral techniques for DNA insertion
• Throughput is low; only a few cells are ‘induced’
• Cancer stem cells (CSCs)
o Some cancer may be considered a disease of stem cell regulation
♣ Evidence is accumulating that indicates that tumors can arise from adult stem cells
♣ Normal stem cells have a hierarchy of slowly dividing cells producing more differentiated cells
♣ Cancer cells seem to be organized in the same way
• Cancers of skin, intestine, blood are very frequent, yet the only cells that are around long enough to accumulate enough mutations are the adult stem cells
o Thought that a stem cell may undergo oncogenic transformation, lose important homeostatic control mechanisms
o Cancer stem cells may be responsible for cancer recurrence in some cases
♣ CSCs appear to repopulate cancers through self-renewal and differentiation of all the tumor cell types
♣ Origin of these CSCs is unclear
o Few cells in tumors are tumorigenic
♣ <1% chance that random tumor cell will generate a new tumor when transferred
♣ Thought that few tumorigenic cells are cancer stem cells
o Cancer Therapies
♣
Current therapies may promote CSC survival and propagation
• Radiation and many chemotherapies target rapidly dividing cells, BUT CSCs would replicate infrequently Thought therapies kill off the bulk of the tumor but NOT the cancer stem cells
• SCs and CSCs are naturally resistant to chemo agents as they have ABC transporters that pump out the drugs (so that the stem cells won’t accumulate mutations) only the susceptible cells die and the resistant cells live
o Ex: paclitaxen in ovarian cancer
o
Prevailing hypothesis is that recurrent cancers are largely the consequence of CSCs slowly repopulating the area
Session 106: Genetic Modifications in Medicine
• Genetically Modified Organisms (GMOs)
o Genome of an organism is modified by genetic engineering techniques
o Routinely used:
♣ Pharmaceutically important drugs (insulin, growth hormone)
♣ Agriculture to enhance the resistance, storage, taste, amount of food products
♣ Environment (clean up spills, ‘sterile’ mosquitoes)
♣ Research purposes
♣ Fun – GloFish, GFP Axolotls!
• Transgenic Mice
o Have ‘foreign’ gene introduced into their genomes – knockin
o Used to study ‘normal’ gene function in mammals and to model human diseases
o The inserted DNA (transgene) usually confers gain of function of that gene
♣ Random insertion of gene, doesn’t typically disrupt genes
o Typically done by pronuclear injection
o Gene is inserted into an Expression vector, typically a bacterial plasmid
♣ Requires promoter, multiple cloning site (MSC) with restriction sites where gene can be inserted, region that encodes a peptide that can be recognized by an antibody, resistance genes for screening purposes (such as ampicillin resistance marker for bacteria)
o Pronuclear injection
♣ Expression vector injected into male pronucleus via non-homologous recombination, then egg is transferred into foster mother
♣ About 10-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line, then can breed mice
o Advantages:
♣ Quick and easy, transgene usually not lethal so some phenotype will be observed
♣ Used extensively for GMOs
o Disadvantages:
♣ Tegulation of the transgene is usually not normal, there can be ‘dosage’ effects because of the multiple gene copies
♣ Endogenous gene is still active so looking for an effect ‘on top’ of the endogenous gene
♣ Transgene protein must be identified separately from the endogenous protein
• Null Mice
o A knockout is the germ line deletion of a specific gene
o Advantages:
♣ Very useful for exploring functions of genes as it assesses what happens in an organism when a gene is missing
♣ Very useful to study development
o Disadvantages:
♣ Interpretation can be complicated by ‘compensatory’ increases in other genes
♣ Deletion may be lethal, slow and a lot of work (and very expensive!)
o Creating gene knockouts in mice
♣ Gene introduced by homologous recombination such that the endogenous gene is removed and a new one is inserted – done is ES cells growing in culture
♣ ES colony with knock out injected into early embryo, which is then implanted into foster mother
♣ Breed heterozygous offspring to get the null animals and hope its not lethal
o Creation of ‘conditonal’ knockouts
♣ Gene can be disrupted only in a specific tissue or at a specific time in development
♣ Most commonly uses the Cre-Lox system
•
Target gene replaced by the same gene that is flanked by the LoxP sites, which are recognized by the Cre recombinase recombinase enzyme then does site-specific recombination using the LoxP sites
• Lox mouse in then mated with a Cre mouse that has the recombinase under the control of a tissue-specific promoter or one that can be induced
• Ex: Estrogen receptor that is sensitive to tamoxifen, the Cre-Lox mouse can be injected with tamoxifen at any time, recombination is induced to remove the targeted gene, effects of the loss can be determined
• Human gene therapy
o Requires the same site-specific knockin and knockout technologies or analogous methods
o Germline human gene therapy – illegal at this time
o Somatic human gene therapy: therapeutic genes are transferred into the somatic cells of a patient
♣ Becoming increasing useful, but not fulfilled expectations
♣ Problems limiting somatic gene therapy
• Vectors often have viral particles, leading to potential problems with toxicity, immune and inflammatory responses
• Corrections are often transient (gene methylation?)
• Chances of inducing tumors
• Regulation and delivery are difficult
• Cloning
o A ‘clone’ is a set of individuals that are genetically identical because they descended from a common ancestor
o Human cloning refers to making an identical copy of a human individual
• Human reproductive vs. therapeutic cloning
o Reproductive cloning (SCNT): embryo generated is implanted into the uterus of a foster mother to create a new individual (could also use iPSCs to get embryo) human cloning
♣ Only require the genome of one individual, a form of asexual reproduction
♣ Currently unsafe with about 95% of cloning attempts ending in miscarriages, stillbirths, etc
♣ Cloned individuals are often biologically damaged
♣ In the future, could replace of cherished loved one OR provide children for those who are sterile or homosexual
o Therapeutic cloning (SCNT): embryo generated is used as a source of ESCs cell or tissue regeneration
Session 107: Intro to Development and Disease
• Basic components of development
o Increase is number of cells and size of organism
o Increase in complexity with diverse cell types
o Patterning (blueprint) and morphogenesis (construction)
o Still occurs after birth
o Influenced by both genes and the environment
• Animal models used to study development (for ethical reasons)
o Experimental models (develop outside mother) – xenopus, chick
o Gentic models: mouse and zebra fish (vertebrates), c elegans and drosophila (invertebrates)
o Much of what we know about development is universal
♣ 3 common germ layers: endoderm, mesoderm, ectoderm
♣ 50% of human genes are conserved in c elegans and drosophila
• Essential genes in embryonic development (first found in drosophila)
• Pair rule genes are transcription factors
o Vertebrate Pax genes – expressed in segments
♣ Mutant Pax1: undulated mouse with shortened vertebral column
♣ Pax3 homozygous mutants (splotch mouse) show spina bifida, brain/neural crest defects
♣ Pax3 heterozygous similar to splotch mouse, pigment defects
♣
Waardenburg’s syndrome patients have mutations in the human homologue of the Pax3 paired box gene allowed sequencing of human Pax 3 analog
♣ Synteny: conserved organization of human and mouse chromosomes
• Regulating expression of different genes in time and space gives rise to diverse cells types
o Regulated expression at transcriptional, post-transcriptional level, or post-translational level
♣ Transcriptional – transcription factors (enhancers, etc), chromatin remodeling
♣ Post-transcriptional level – RNA processing, inhibitory proteins, miRNA
♣ Post-translational level – phoshorylation state, nuclear vs. cytoplasmic
o Regulation of master transcription factors and hox transcription factors
• Master transcription factors – Cell specification/fate
o Cell face specification: Ex. Fat cell vs. muscle cell
o Pax-6
♣ In drosophila, artificial expression in leg gives ectopic eye on leg
♣ Can induce undifferentiated cells into an eye, required for eye formation
♣
In humans, aniridia is caused by mutation in the pax-6 gene (black iris, poor vision)
♣ Transcription factor, contains conserved motif for DNA binding (pax-1, pax-3, pax-6)
• Paired box and paired homeobox
• Mutation in these causes impaired function – required for DNA binding
o myoD – master transcription factor for muscle
♣ Fibroblast myoD muscle cell
• Hox genes – specification of cell identity (brain vs. spinal cord, what KIND of neuron) along anterior-posterior axis
o Transcription factors that have a homeobox DNA binding region
o Found in a cluster along the same region of the chromosome
o Position of the genes in the homeotic complex corresponds to body segment
o Mutations in Hox genes cause homeotic transformation
o
Humans have multiple redundant copies of Hox genes, but can still have homeotic transformations
♣ Ex: Hoxb-2 expressed from shoulder to legs, Hoxb-4 expressed from arms down
♣ Hoxb-4 knockout mouse: C2 vertebra transformed into more anterior C1
o Hox genes and human disease
♣ HoxD13: synpolydactyly – short, fused fingers
o Retinoic Acid is a teratogen – caused misexpression of hox genes
♣ Causes homeotic transformation in the hind brain
♣ Causes more anterior expression of Hoxb-1 in hind brain rhomboid 2/3 intro rhomboid 4/5
o How is A/P pattern of Hox gene expression set up?
♣ miRNAs play an important role in where Hox genes are expressed
• miRNA-10 targets Hoxb4, miRNA-196 targets Hoxb8
• Wherever these miRNAs are expressed, you get targeted destruction, silencing
Session 108: Spatial and Temporal Signaling in Development
• Induction – inducer, responder, competence
o A process by which one population of cells (inducer) affects the development of another (responder) through signaling
o Two types
♣ Paracrine – involve diffusible molecules
♣ Juxtacrine – involve cell contact
o Example: Eye development
♣ Normal induction of lens by optic vesicle (inducer)
♣ Only ectoderm in head region is competent to receive inducer signal and respond
♣ Optic vesicle has high expression levels of FGF8 inducing factor
♣ Head ectoderm expresses pax-6, required for ectoderm to be competent for lens induction
• Most embryonic inductions are mediated by secreted signaling factors
o Can act on remote cells in paracrine fashion
o Can form gradient, affecting cells differently depending on the concentration of the signaling factors
o Always activate intracellular signaling pathways
•
Four key signaling pathways in development - involved in INDUCTION
o FGF pathway – binds to tyrosine kinase receptor, activates MAP kinase pathway; mutations typically affect bone development
♣ BMP7: kidney and eye development, skeletal patterning
♣ BMP2: heart development
♣ BMP8: spermatogenesis
o Hedgehog (Hh) – membrane receptor Patched stops inhibition of Smoothened Gii enters nucleus transcription, Gli is either an activator or a repressor
o Wnt pathway – Frizzled Disheveled inhibits GSK-3 b-catenin not degraded
o TGFb – Smads (see previous)
♣
Associated with diseases of limb formation
• Sequential inductive interactions lead to pattern formation
o Specification of embryonic axis
♣ A/P axis
♣ D/V axis
♣ L/R axis
o Eye development
♣ Optic vesicle induces formation of optic placode lens placode induces formation of optic cup lens capsule induces formation of cornea
o Neural Tube Patterning – dorsal/ventral patterning
♣
Two signaling pathways – sonic hedgehog (notocord) and BMPs (ectoderm)
♣ Ventral patterning of neural tube induced by Shh secreted by notocord (HEDGEHOG signaling pathway) – D/V concentration gradient
• High concentration in floor plate motor neurons
• Lower concentration towards roof plate different types of interneurons
♣ Dorsal patterning of neural tube induced by BMP4 and 7 secreted by epidermis and roof plate turns on TGFb signaling pathway
• High concetration in roof
♣ Gradients of the two paracrine factors on opposite ends of D/V axis results in production of different transcription factors, which specify different neuronal cell fates
• Hedgehog signal transduction pathway mutations
o Gli truncation leads to Pallister-Hall syndrome
♣ Gli is continually active as a repressor
♣ Lots of digits – no proper patterning
o Greig cephalopolysyndactylyl is due to loss of function mutation of Gli
♣ Gli cannot act as an activator or a repressor
♣ Megalocephaly, broad thumb, duplicated big toe – duplicated fused digits
o Mutations in patched receptor lead to Gorlin’s syndrome/basal cell syndrome
♣ Overactivation of Hh signaling due to constitually active Smoothened signaling even without hedgehog ligand (Patched normally inhibits Smoothened in absense of Shh ligand)
♣ Cancer due to overproliferation of cells in skin, eye
o Loss of hedgehog molecule leads to holoprosencephaly cyclopia
♣ Major developmental disorder
♣ Affects midline patterning
♣ Can be as mild as only having a single front tooth to having a single eye (cyclopia)
♣ Can also be caused by defects in the synthesis of cholesterol
• Hh requires cholesterol modification to create smooth Hh gradient through signal diffusion
• Jervine alkaloid inhibits cholesterol synthesis causes similar symptoms
• FGF signaling pathway mutations – generally affect bone formation and limb development
o Crouson’s, Pfeiffer, and Apert Syndromes
o Achondroplasia – congenital dwarfism
• Wnt Signaling and Disease – generally gives rise to cancer
o DES (diethylstilbestrol) – teratogen causing abnormal reproductive tract in fetus
♣
DES inhibits production of Wnt7a in epithelial tissue
♣ Loss of Wnt7a signaling means Hox and Wnt5a are not induced in mesenchyme
o APC mutations lead to FAP hereditary colon cancer
Session 109: Mechanics of Morphogenesis and Cell Adhesions
• Morphogenesis
o How are tissues formed from populations, how do they migrate to the correct layer, etc…
• Three germs layers
o Holfreter’s experiment to show cells sort themselves
o Reconstruction of dissociated skinc ells from 15 day mouse embryos
• Cell adhesion as a mechanism of morphogenesis
o Generates boundaries between different cell types
o Formation of tissues from individual cells, and maintenance of tissue integrity
o Formation of organs and maintenance of organ integrity
o Establish connections between different cell types that need to communicate (neurons and mucle)
•
Cell adhesion molecules
o Cadherins – large family (desmosomes, adherent junctions)
♣ Classic cadherins: E-cadherin, P-carherin, N-cadherin
♣ Homophilic adhesion
♣ Injection of cadherin mRNA in xenopus embryo results in loss of cell adhesion
♣ N-cardherin establishes boundary between neural and epidermal ectoderm
♣ To form adhesion, Ca2+ must be in solution and must be associate with actin cytoskeleton
• Associated with cytoskeleton by catenins (remember Wnt pathway???)
♣ Signaling pathways and disease (crosstalk)
• Wnt signaling pathway b-catenin: colon carcinomas, melanomas probably due to loss of adhesion, contact inhibition through crosstalk with Wnt signaling pathway
♣ Diseases associated with cadherins – normally loss of classical cadherins leads to death of embryo due to importance in embryogenesis
• E-cadherin: tumor malignancy (loss of contact inhibition cell proliferation)
• Demosomal cadherin: pemphigus vulgaris (autoimmune disease, Ab made to desmonsomal cadherin)
o Skin, mucous membrane blistering due to loss of desmosomes in stratum spinosum layer of skin
• Cadherin 23, protocadherin 15: usher syndrome
o Common form of hereditary deafness – disorganization of hair cells due to loss of stereocilia connection by cadherin molecules
o Retinitis pigmentosa
o IgCAMs – very large family
♣ Calcium independent
♣ Extracellular globular domains held together by disulfide bonds
♣ Mediate homophilic and heterophilic binding with other molecules, including extracellular matrix (collagen, proteoglycans, fibronectin, laminin)
♣ Weaker adhesions than cadherins
♣ Generally thought to be of importance for transient cell adhesion
♣ Generally found in neuronal cells (NCAM) important in axon guidance and neurite growth
♣ Associated Diseases
• CRASH and L1 (IgCAM linked to actin cytoskeleton by ankyrin)
o Corpus callosum hypoplasia
o Retardation, mental
o
Adducted thumbs
o Spastic paraplegia
o Hydrocephalus
• Autism, Schizophrenia, Cancers
o Integrins – large family (hemidesmosomes)
♣ Heterodimeric proteins composed of one alpha and one beta subunit
♣ Cell-cell (typically heterodimeric) and cell-extracellular matrix (ECM) adhesion
• Links EXTRACELLULAR matrix (via talin, viculin, alpha-actinin) and INTRACELLULAR actin cytokeleton
♣ Dependent of extracellular divalent cations (Ca, Mg) for ECM binding
♣ Much weaker than cadherins, but typically present in high concentrations (like Velcro)
♣ Important in cell migration
♣ Associated Diseases
• Angiogenesis, Inflammatory diseases, cancer, myopathy
• Epidermolysis Bullosa
o Skin blister disease, may be fatal
o Defect in cell adhesion, but defect in hemidesmosomes in basal layer of skin that connect cells to basement membrane
♣ Play important role in cell migration – migration of neural crest cels, migration of blood cell precursors to liver, bone marrow, etc…
• Cell Adhesion are important
o Tissue boundaries, cell sorting
o Epithelial adherens junctions maintain tight protective layer
o Cell migration, neural crest cells must migrate, requires cell adhesion molecules
o Axon guidance, synapse targeting, and adherens (connection between neurons, muscle cells)
• Types of cell adhesion
o Homophilic binding – one adhesion molecule binds to same molecule on other cell
o Heterophilic binding – adhesion molecule binds to different molecule on other cell
• Cell migration as a mechanism of morphogenesis – extension, attachment, translocation, de-adhesion
o Migration of cells during gastrulation
♣ Migrating ectoderm forms endoderm and mesoderm
o Neural crest cells undergo extensive migration
♣ Neuroectoderm cells that for neurons, Schwann cells, pigment cells
♣ Defects can give piebaldism, where pigment is missing from forehead, stomach
♣ Hirschspring’s Disease – congenital constipation of lower bowels caused by absence of ganglia that regulate preristalsis
o How cells migrate
♣ Cells extent filopodia, lamellipodia and form contacts with substratum
♣ This connected in mediated by integrins
• Helps create force/traction so cells can move against substratum
o How do cells decide where to migrate?
♣ Haplotaxis: migration based on changes in adhesiveness of the substratum
♣ Specific substrate guidance: migration along a pathway made of a specific substance (eg laminin, collagen) – sensory neuron grows processes onto laminin but not collagen
♣ Chemotaxis: migration regulated by a gradient of diffusible substances sensed by the cell via cell surface receptors (like bread crumbs) – neutrophil migration
o Example: directing axon migration in neural tube
♣ Floorplate neurons must migrate ventrally, then anteriorally
o Chemotactic cues can be attractive and/or repulsive
♣ Attractive: N-formylated peptides produced by bacteria attract neutrophils which sense cue via a cell surface receptor
♣ Repulsive: Slit repels axons expressing the Slit receptor, Roundabout (Robo)
• Slit found at midline, so axons/neurons expressing Robo won’t be seen at midline
• Slit knockout axons with Robo found at midline
♣ Both: Netrins are secreted by floorplate on the central part of neural tube, direction the ventral migration of some axons and repelling migration of others
• Some neurons are attracted to Netrins, some are repelled
• Why? Different neurons express different receptors type of guidance molecule and receptor
Session 110: Human Malformations and Teratogenesis
• General
o Typically occur in the first 12 weeks of gestation – period of organogenesis
o Birth defects are common: 1/30 babies are born with a ‘birth defect’
o 5th leading cause of death in children 5-14 years
o 4th leading cause of infant mortality worldwide
o Causes
♣ 95% genetic – complex of multifactorial 50%
♣ 5% environment – infections, prescription drugs, recreational drugs/ethanol, method of conception
• Genetic Malformations
o Chromosomal basis of birth defects
♣ Standard chromosome studies will be abnormal in about 4% of infants with birth defects
♣ With array CGH up to 20% will be identified to have a significant chromosome abnormality
o Single Gene Disorders
♣ Although there are many single gene disorders that cause birth defects, there are no current means for screening multiple genes at once
♣ Testing relies of ability of clinician to recognize pattern of malformations – syndrome
♣ Syndrome Recognition
• Clinical recognition of a constellation of findings that when identified together in a patient constitute a known condition
• We do not known the exact cause for the majority of birth defects - Multifactorial
o The ‘complex’ or ‘multifactorial’ model is used to explain the majority of birth defects
o Most common defects – congenital heart defects, cleft lip and palate, neural tube defects
o Neural tube defects
♣ 1930’s – noted that women with good nutrition had lower risk
♣ 1980’s – found folic acid was protective, confirmed in early 90’s through case control studies
♣ Folic acid is now given prenatally to women to prevent NTD
♣ Cereals and grains are now fortified with folic acid
♣ Rates have decreased since fortification with folic acid
♣ Hypothesis: since folic acid lowers the risk of NTD, then the genetic cause may lie in genes that encode proteins or enzymes that effect folate metabolism
• Found that MTHFR polymorphism T/T homozygotes (10% of population) increase risk – homozygous mothers have relative risk of 1.6
♣ May be gene/ environment interactions
o Cleft lip and palate
♣ Like NTD, there is an increased risk of cleft lip in palate in subsequent pregnancies (5-7% risk of recurrence)
♣ Complex inheritance model
♣ Multiple studies have shown an increased risk of cleft lip/palate to mothers who smoke – odds ratio of 1.3
• Environmental causes of birth defects - teratogens
o Timing
♣ There are critical periods in embryonic periods where embryo is most susceptible
o Alcohol – 30-60% fetuses affected
♣ Recognizable pattern of facial findings (short nose, flattened midface, thin upper lip, etc), growth delay, heart defects, cognitive disability and behavioral difficulties
♣ Later exposure results in cognitive disability and behavioral difficulties without facial findings
♣ There is no known safe amount of alcohol during pregnancy
♣ Annual costs related to caring for ethanol exposed infannts is in the BILLIONS
o Prescription medications
♣ Isotretinoin – 30%
♣ Thalidomide – 10%
• Associated with severe malformations of their arms and legs
• Thalidomide was used as a sedative to help pregnant women remain ‘calm’
♣ Warfarin – 8%
♣ Diazepam – 1%
o Maternal Infections
♣ Cytomegalovirus (CMV)
• 1% of infants are infected with CMV at birth
• 10% of infected infants can have sequelae
o Hearing loss, microcephaly, brain malformations, rash
♣ Rubella
• Common prior to universal immunization in the 1960s
• Concerns with re-emergence in area with low immunization compliance
• 90% of women infected during the first trimester will have infected offspring
• Clinical Findings
o Cataracts, deafness, brain malformations, microcephaly, rash (blueberry muffin baby)
o Assisted Reproduction
♣ 10% of couples experience infertility
♣ Now ask mode of conception as part of history taking in genetics clinic
♣ In vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSE)
• Higher incidence of imprinting disorders (especially maternal imprinting): Angelman, Beckwith-Wiedmann
• Higher incidence of cleft lip/palate, hypospadias, esophageal atresia, imperforate anus
Session 111: Apoptosis in Development
• Apoptosis is genetically programmed
o Apoptosis is programmed cell death – genetically controlled
♣ Cell membrane is intact, no cytoplasm leaks out
♣ Genes are turned on to induce morphological changes – degradation of cell contents
♣ Chromatin compaction gene causes DNA condensation and cleavage
o Necrosis is cells that are damaged by injury or exposure to toxic chemical, cells swell, lyse, cause inflammation of surrounding tissues
• Apoptosis in tissue morphogenesis
o Sculpting tissue/organ
♣ Fingers, limb development
♣ Tube formation (hallowing out)
♣ Bone formation – hypertrophy and apoptosis of chondrocytes to for bone
o Deletion of unwanted structures
♣ Vestigial structures removed – like tadpole’s tail
♣ Tissue homeostasis – mammary secretory epithelial cells that increase during lactation die after weaning
♣ Development of male and female reproductive organs (Mullerian and Wolffian duct)
o Regulation of number of cells
♣ Number of motor neurons are determined by size of target tissue to be innervated, neurons that don’t ‘make it’ undergo apoptosis
o Elimination of damaged or harmful death
♣ 95% of thymocytes generated in thymus (self-reactive) die
♣ Cells that have incurred DNA damage
o Production of specialized cells like lens epithelial cells, keratinocytes, RBCs
♣ Lens cells are actually dead – apoptosis of nucleus occurs but cell is not phagocytosed
•
Caspase cascade
• Apoptosome
• Suicide (intrinsic) pathway via mitochondria
o First discovered in c elegans
o Induction of procaspase activation
o Caspase-9 – protease, induces caspase-3 (caspase cascade)
o Caspase Cascade:
♣ Release of cyt c binds to Apaf1 causes conformational change forms apoptosome recruitment, cleavage of procaspase-9 active caspase activates other caspases (caspase-3) cleavage of cytosolic proteins, nuclear lamins, etc…
o Bcl2 proteins
♣
Anti-apoptotic proteins (Bcl2, Bcl-Xl) – 4 BH domains
• Binds to pro-apoptotic BH123 proteins to block pore formation cyt c cannot escape for mitochondria
♣
Pro-apoptotic BH123 proteins – 3 BH domains (Bax)
• Found in mitochondrial membrane
• Aggregation causes formation of pores to release cyt c caspase cascade
♣ Pro-apoptotic BH3-only protein – 1 BH domain (Bid, Bad?) – sequester Bcl2 away from Bax channel
o Expression of Bcl-2 is dependent on the Mift transcription factor
♣ Mift is activated by MAP kinase cascade transcribes genes that ensures cell survival of melanocytes
♣ Piebaldism results in loss of melanocytes regulated by the Mift transctiption factor
o Bcl-Xl expression controlled by erythropoietin
♣ Bcl-X regulates how many red blood cells go into circulation
♣ Binding of erythropoietin to receptor causes downstream transcription of Bcl-Xl anti-apoptotic protein more RBCs survive
• Signals inducing apoptosis
o Loss of trophic factors
♣ In presence of survival factors, Bcl2 production is increase apoptosis blocked; inactivation of pro-apoptotic BH3-only Bcls protein
♣ Absense of survival/trophic factors promote apoptosis
• Activation of caspase cascade
o Damaged cells via p53
♣ PUMA (p53 Upregulated Modulator of Apoptosis)
• Pro-apoptotic Bcl2 protein – contains BH3 domain, binds to anti-apoptotic Bcl2 proteins
• Murder (extrinsic) pathway receptor mediated
o Generally induced by cytotoxic T cells
o T-cell receptor binds to histocompatability molecule produced FasL ligand binds to Fas receptor
on target cell (ALL cells express Fas receptor)
o Causes trimerization of receptors death domains aggregate (allows formation of DISC) recruits and actvates caspase 8 more caspases recruited (caspase 3)
o Mutations in FasL or FasR or caspases in pathway, have defective apoptosis
o FasL and immune privileged sites
♣ Very little immune response in brain, eye, etc
♣ Have physical structures AND immune blockers
♣ Eye cells have FasL that is constitually active induces apoptosis in immune cells that enter the eye
♣ In these tissues, immune response (inflammation) would cause a lot of damage
• Homeostasis – balance between cell division and cell death
o Too much cell proliferation causes cancer, SLE, rheumatoid arthritis, polycythemie
o Too much cell death causes neurodegenerative diseases (huntington’s, ALS), autoimmune diseases (AIDS), stroke, MI
Session 112: Organogenesis
• How the endoderm gives rise to the digestive and respiratory tracts
o Tissue becomes specified to a certain organ, then cell differentiation occurs
• Gut development
o
Differentiation into foregut, midgut, hindgut is caused by relative levels of Wnt, Fgf4, BMP signaling molecules
o Different parts of gut express different transcription factors (see figure)
o Know Pdx1 required for pancreas formation
o As gut invaginates, Sonic Hedgehog (Shh) is expressed in hindgut first turns on expression of specific Hox genes
o Paracrine signals from cardiogenic mesoderm (FGF)
♣ Highest levels received by lungs, then liver
o Liver formation
♣ Receives FGF signal from cariogenic mesoderm
♣ Liver endoderm made competent to receive signal by BMPs
♣ Induces formation of hepatocytes, begins expressing Prox1 allows cell proliferation
• Reduces levels of E-cadherin
• Proliferating cells need space, start proliferating into mesenchyme (which produces hepatocyte growth factors)
• Prox1 mouse mutant: no cell proliferation, no cell migration, high levels of E-cad
o Pancreas formation
♣ 2 buds formed one next to liver (ventral bud), one dorsal bud
♣ Eventually migrate and fuse
♣ Ventral endoderm
• Low FGF, BMP, but does express PDX1 (master transcription factor for pancreas)
• Migrates to dorsal bud, where they fuse
♣ Dorsal endoderm
• Also expresses PDX1
• Signaling from notocord, expresses FGF2 and activin
• Induces endoderm to turn down Shh induces pancreas formation
o Forcing Shh expression means dorsal pancreas doesn’t form
♣ Pdx1 transcription factor
• Master transcription factor
• Induces budding from the gut epithelium
• Represses gene expression characteristic of gut tube other than pancreas region
• Maintains repression of Shh in pancreas region of gut
♣ Pdx1 and disease
• Loss of Pdx1 results in loss of pancreas formation
• Humans homozygous for Pdx1 mutation do NOT develop a pancreas die
• Patients heterozygous for Pdx1 mutation develop MODY4 diabetes (Pdx1 is also important in beta cell formation)
• Lung formation
o Tbx4 helps tracheoesophogeal folds to fuse
♣ Loss of tbx4 leads to tracheoesophogeal fistula
o Mesenchymal interactions required for bronchial branching
♣ Fgf10 found in mesenchymal cells acts as a chemoattractant for lung epitherium
♣
Tips of lung buds express Fgf receptor that responds to signal migrate and grow towards cells expressing Fgf10
♣ Lung bug epithelium exposed to Fgf10 expresses sonic hedgehog (Shh) this signal diffuses to mesenchymal cells, which stop Fgf10 production
♣ This leads to BRANCHING
• Epithelium-mesenchymal interaction for organ fine tuning (found in many different tissues)
o
All organs have an epithelium (one of three germ layers) and mesenchyme (mesoderm or neural crest)
o Epithelial-mesenchymal inductions display regional specificity; mesenchyme specifies the structure to be formed
♣ In chick embryo, mesenchyme from wing, thigh, and foot transplanted with wing epidermal epithelium induces formation of wing feather, thigh feather, or scales, respectively
♣ Epithelium is a ‘blank slate’ induced to form different structures by mesenchyme
Session 113, 115: Limb Development
• Limb development is the most well understood organ development
o Malformed limbs are not lethal, unlike malformation of many other organs
o
Chick wing development is very similar to human limb development
• Initiation (where limb growth starts)
o What determines where limbs are formed? Hox genes (important in patterning along A/P axis)
♣ Hox genes specify region of the embryo, then master transcription factors are turned on
♣ Most anterior region where HoxC-6 is not expressed is where forelimb develops
♣ Most posterior region where there is no HoxC-8 expressed is where hindlimb develops
o
What induces limb bud formation?
♣ High expression of FGF10 in mesoderm where limb bud develops
♣ First expressed uniformly, then localized to where limbs develop (probably based on Hox genes)
♣ Induces epithelium (apical ectodermal ridge) to express Wnt3a FGF8 signals back to mesoderm to keep expressing FGF10
♣ Ectopic FGF10 can induce additional limb formation
o What specifies fore vs. hindlimbs?
♣ Tbx5 (forelimb) and Tbx4 (hindlimb) are important transcription factors
♣ Ectopic FGF10 between fore and hindlimb induces formation of chimera limb that expresses both Tbx5 and Tbx4
♣ Clinical: Holt-Oram syndrome
• Patients are heterozygous for mutations in Tbx5
• Also known as heart-hand syndrome
• Heart and upper limb malformations: absent thumbs, distally placed, duplicated, or triphalangeal thumbs
• Partial or total absence of forearm
• Proximodistal axis: FGF pathway
o Mesenchymal cells hold a lot of information, epithelium is a ‘blank canvas’
♣ ‘Progress zone’ (regional of mesenchymal cells) that undergoes proliferation
♣ Forms all cells of limb
♣ Apical ectodermal ridge responds to FGF signaling from ‘progress zone’ (see above)
• Produces Wnt3a and FGF8
• FGF8 is required to maintain cell proliferation in progress zone (also maintains FGF10 expression) but contains no positional information
♣ Mesenchyme induces and sustains AER and specifies type of limb; AER sustains outgrowth and development of limb and directs P/D growth
♣ AER only maintains proliferation of progress zone – implantation of old AER into young limb bud results in a normal limb (vice versa with young AER)
♣ Positional information lies with mesenchyme (progress zone) – implantation of old limb bud onto young limb bud results in only distal structures
o
How does mesenchyme specify the proximal-distal axis?
♣ Progress zone model: time - how much time is spent going through cell division determines what kind of structure develops (proximal vs distal)
♣ Early allocation and progenitor expansion model: space – there are four regions of limb bud, each region just expands in size as limb grows (cell fate is specified from the beginning to become proximal vs distal stuctures)
o Hox (A and D) genes specify the P/D regions of the limb (cell fate)
♣ Mutations in HoxD13 give mutations in most distal part of arm (digit formation)
♣
Deletion removing HoxD cluster leads to severe developmental defects in distal regions (11, 12, 13 ulna, metacarpals, digits)
•
Anteroposterior axis: SHH pathway
o Anterior = thumb; posterior = pinky
o Zone of polarizing activity (ZPA) – region of the limb mesenchyme in the posterior limb bud
♣ Required for A/P patterning of the limb
♣ Secretes sonic hedgehog (SHH) (induced by FGF8 secreted by AER)
• Increases activator Gli3:repressor Gli3 ratio with the highest levels of activator in the posterior
• Initiates and sustains a gradient of BMP (2, 7 - TGFb family) signals in interdigital mesoderm specifies digit identity
• BMPs induced in webbing between digits specify digit identity
o Clinical: SHH misexpression and congenital limb defect
♣ SHH contains enhancer far away (in different gene); important for direction expression of SHH
♣ Mutations in enhancer lead to limb malformation extra digits, mirror imaged limbs
♣ Gli-3: Grieg cephalopolysyndactyly and Pallister-Hall syndrome with polydactyl, abnormal facial, cranial formation
• Dorsoventral axis: Wnt pathway
o Dependent on surface ectoderm
o Wnt7a is expressed in the dorsal but not ventral limb ectoderm
o 180 degree rotation of ectoderm partially reverses polarity on distal structures
o Lmx1 transcription factor, activated by Wnt7a
♣ Specifies dorsal identity in limbs
♣ Expressed only in the dorsal limb mesenchyme
♣ Ectopic expression of Lmx1 in ventral limb mesenchyme induces dorsal phenotype
♣ Lmx1 mutant mouse knockout results in mice with no dorsal limb
♣ Clinical: Nail-patella syndrome – dominant disorder resulting from mutations in Lmx1 gene
• Dorsal tissues are partially ventralized
•
Coordinating three axis
o Fgf8 is required to induce Shh expression, thus coupling A/P patterning to P/D growth
o Wnt7a is required to maintain Shh expression, coupling A/P patterning to D/V
♣ Knocking out Wnt results in abnormalities of D/V patterning and results in loss of 5th digit as well
o AER forms in response to FGF10 only where dorsal and ventral ectoderm are juxtaposed, coupling P/D growth to D/V patterning
• Morphogenesis – adhesion, apoptosis, migration; occurs after patterning, limb bud formation
o Sculpting the autopod (hand)
♣ Cell death is required
♣ Apoptosis occurs in intidigital regions, as well as space between radius and ulna
♣
BMPs are important for inducing cell death
• Inhibition of BMPs (by noggin) maintains interdigital tissue
• Also involved in inducing chondrocyte formation
• Note: BMP turns on FGF pathway
♣ TGFb/BMP signaling and disease (loss of GDNF?)
• Symphalangism – loss of second phalanges
• Brachydacyly – fusion of joints (no cartilage formation)
o Bone formation
♣ Bones must undergo apoptosis and chondrocyte formation
♣ Apoptosis shapes bone
• FGF and Disease
o Directs chondrocyte formation, and thus is important in bone formation
o Mutations give Pfeiffer syndrome (early closure of sutures, very severe), Crouzon syndrome, Apert Syndrome (includes syndactyly), achondroplasia (short limbs due to disturbed growth)
o Activation of FGF receptor is constitutive after mutation leads to p21 activation brings cells out of cell cycle (Cdk inhibitor) leads to cartilage growth stopping early stunting of growth
Session 116: Pharmacogenomics
• What happens to a drug when it enters the body?
o Absorption
o Distribution
o Metabolism
♣ Biochemical modification, xenobiotic metabolism often converts lipophilic chemicals to more readily excreted polar products (p450s)
♣ Metabolism can result in activation or deactivation of drug
o Excretion
o ADME process – each step is affected by genetic differences in people
♣
Receptors, ion channels, transport molecules, signaling pathways, metabolic pathways – all part of GENETIC PROGRAM of an individual
• Pharmacokinetics
o Time course of drug and metabolite levels in different fluids, tissues, extreta of body
o Different for different individuals
o Drug dose can be represented as the area under curve (AUC)
o Therapeutic window – amount of medication between the amount that gives the effective dose and the amount that gives more adverse effects (toxicities) than desired effeects
• Pharmacodynamics
o Biochemical and physiological disposition of the drug within the body (often related to receptor interactions and transport)
• Example: Cetuximab
o An EGFR inhibitor, given by IV for treatment of metastatic colorectal cancer, head/neck cancer
o About 75% of metastatic colorectal cancers have EGFR+
o BUT…40% do not respond to Cetuximab – have an activating mutation in the RAS gene
♣ EGFR signaling is not required to activate RAS pathway
o Genetic testing recommended to look for mutations in RAS before prescribing Cetuximab
• Pharmacogenomics
o Using what we know about genetic variations in individuals to predict drug response in an individual
o FDA currently has about 80 recommended companion genetic diagnostics with drugs
• Drug metabolism
o Phase I Enzymes: add or expose polar groups to inactivate enzymes or activate proenzymes by oxidation, reduction, hydrolysis
o Phase II Enzymes: conjugate other molecules to drug (methylation, sulphation, acetylation, glucuronidation) to increase mass of drug most often inactivate drug
• Drug dose and response is related to genetic variations
o Ex: Prozac overdose in 9 year old child
♣ There are at least four genetic variations that effect Cyp 2D6
♣ Variations cause differences in metabolism (poor intermediate extensive utra)
♣ Can either cause reduced ability to clear or activate drugs or increased activity accelerating clearance or activation
♣ 7% of Caucasians are poor metabilizers vs <1% of Asians
• Frequency of variations is different in people of different ethnic backgrounds
• Also is more common in redheads linkage disequilibrium with Cyp 2D6
o Cyp 2D6 affects metabolism of many psychiatric drugs important to test for Cyp 2D6 genetics to find the ideal dose for each individual
o NOW, dose adjustment is typically done with trial and error, in the FUTURE, genetics will probably be used to find a more ideal dose from the outset personalized medicine (family history, clinical data, genomic profile all used together)
o Ex: Tamoxifen used to treat ER+ breast cancer
♣ Cyp 2D6 metabolizes prodrug tamoxifen to MUCH more effective endoxifen
♣ Those with ineffective Cyp 2D6 activity do not effectively convert prodrug to active drug
♣ IDEA: if cancer patient isn’t experiencing any side effects, must think about genetic testing to see if patient has appropriate metabolic activity to activate drug
• Health care impact of genomics
o Pre-genomic era: disease description, uniform disease classification, patient homogeneity, universal therapy
o Post-genomic era: disease mechanisms, disease heterogeneity, individual variability, targeted therapies
• Genome testing issues
o Privacy and confidentiality, stigmatization as ‘untreatable’, need for new guidelines, incidental findings
o Personal information is not unique to the patient, it also has implications for family members
o GINA: employers, health insurance cannot discriminate based on genetic status, but life insurance, military can discriminate
o Race/ethnicity
♣ Does the emerging data of race/ethnic difference in genetic variations lead to racial profiling in health care delivery?
♣ Many issues with drugs targeted towards certain ethnic groups
• Genetic variations leading to variability in Warfarin response
o Two genes – CYP2C6 and VKORC1 – affect metabolism of Warfarin
o Mayo study found that using genetic information to determine dose led to a 30% decrease in hospitalization costs