Exam 3 - HG (copy)
Genetic Testing & Diagnosis
Test fall into 3 basic categories
Tests before conception: discover what gene an adult has & how it can be passed to their children and discovering what disease gene an embryo may have
After conception: discover genetic diseases in developing fetus
After Birth: diagnosing person who has symptoms
Types of tests
Predictive / presymptomatic screening
Used to detect gene mutation causing disorders after birth or later in life
Can determine chances of developing certain cancers, typically only tells you your risk
Can predict genetic disorder before symptoms develop
Allows patient to help make decisions about future medical care
Types:
1)
Presymptomatic_____________ Testing = positive result means you will develop symptoms of
disease at some time in your life (Huntington’s Disease)
2)
_____Predisposition____________ Testing = positive result means you are more likely to develop
symptoms of disease, but it is not definite (BRCA and breast cancer)
Preimplantation screening
Specific technique for ____reducing the risk____________ of having a child with a particular genetic or chromosomal disorder
_____specifically detect______________ genetic changes in embryos for IVF
Small number of cells taken from embryo and tested, occurs before ___implanation___________
Candidates, tests over 100 genetic conditions
- Sex linked disorders
- Single gene disorders
- Women over 35
- Recurrent pregnancy loss
- Prenatal screening
- Detects changes in fetus genes or chromosomes ___before birth_______
- Typically offered during pregnancy if there is increased risk
- Cannot cover all inherited disorders/birth defects (there are 4,000 that we know of)
- Only lessens parent’s uncertainty or help them make decisions
- Includes:
- 1)____non invasive _________ procedures = (after 8 weeks) Cell free fetal DNA analysis from maternal blood, can reveal sex of fetus & specific genetic abnormalities
- 2)__invasive ________ procedures = chorionic villus sampling (after 10 weeks) & amniocentesis (after 15 weeks
Newborn screening
Testing ____automatically__________ done _____just after birth__________ to identify genetic disorders
Most are for conditions that are serious if treatment not started early in life
32 core conditions every state screens for = most are inborn errors of metabolism __________________________
Varies from state to state (Tennessee screens 70 different conditions)
Amino acid disorders = PKU
Endocrine disorders = adrenal dysfunction too much androgen
Fatty acid oxidation disorders = body cannot break down fatty acids
Hemoglobin disorders = sickle cell
Lysosomal storage disorder = lysosomes cannot break down fats
Organic acid disorders = metabolic condition where acids build up in blood & urine
Other = CF, SCID, Critical Congenital Heart Disease
Diagnostic screening
To ___identify or rule_____________out a specific genetic or chromosomal condition
Used to confirm_____ a diagnosis when signs & symptoms indicated it
Can be performed at ___any stage _______ before or after birth
Influences patients choices about health care and management
Carrier screening
Typically done for those with a family history of a genetic disorder or certain ethnic groups that have an increased risk of a genetic condition
1) targeted____ carrier screening = based on your ethnicity or family history
2) ___expanded_______ carrier screening = tests 100 genes for specific disease that severely affect quality of life, not based on ethnicity or family history
Identifies those who carry ___1 copy ____ of a gene that when present in 2 copies causes a genetic disorder (thus only one of the couple needs to be tested)
Provides information for a couple that are planning on having a child in the future
Will tell the risk of having a child with that particular genetic condition
Forensic screening
Identify person for legal purposes__________
not used_______for mutations associated with disease (no real medical application)
Good for:
- Identify or exonerate a person involved in a
- crime/investigation
- Victims of catastrophe
- Establish biological relationships (paternity)
- Direct to direct consumer tests
(+) Provides information without ________involving doctor or insurance _____________________
(+) Easy: Collect sample at home (buccal swab or blood draw) → mail to laboratory
(-) Can be misled by _____unproven tests__________or person providing skewed results
(-) ___genetic privacy_____________
(-) May need someone to interpret or help make decision
- Cannot tell you
1. If ___you will show_____________________of a disorder
2. ___how severe________ the symptoms will be
3. How the disorder will ____progress over time____________
- More direct to direct consumer tests
23 & Me =
(+) genetic information & ancestral background
(-) genetic mining & pharmaceutical collaborations (Google, Johnson & Johnson)
- Color Genomics = Google, Twitter, Stanford, MIT, Berkely
- fully automated laboratory, analyzes 19 genes of breast & ovarian cancer
- $249/test and provides genetic counselor
- Counsyl = genetic screening service for chromosome conditions, inherited cancers
- Gene by Gene = Whole genome & exome sequencing & mitochondrial testing
- LabCorp of America = diagnostic testing, predictive analysis, personalized medicine
- Myriad Genetics
- Cornered the market in three areas
1. Predictive medicine ( asses risk)
2. Personalized medicine (response to a drug)
3. Measurement of disease activity
- Discovery of BRCA genes
- BRACAnalysis = test for hereditary breast & ovarian cancer
- COLARIS = test for hereditary colorectal and uterine cancer
- MELARIS = test for hereditary melanoma
- TheraGuide 5-FU = personalized medicine for toxicity test to chemotherapy
- Panexia = test for hereditary pancreatic cancer
- Vectra DA = blood test for rheumatoid arthritis
- Supreme court ruling = tried to patent the BRCA gene
- Immune system: overview
Immune system composed of:
- 1.Immune cells (duh!)
- 2.The___molecules________ immune cells secrete
- 3.The____organs/tissues________ where immune cells are made/stored
- The effectiveness of your immune system is to be able to recognize
- 1. Self vs. non-self cells
- 2. Between healthy vs compromised SELF CELLS (viral infected or cancer cells)
- 3. Between healthy vs pathogenic NON -SELF CELLS (lots of beneficial microbes)
- ***can tell the difference between self vs non-self by cell surface Antigens ____
- immune cell surface molecules
- Chromosome 6 = encodes Major Histocompatibility Complex (MHC) that encode Human Leukocyte Antigens (HLA)
- 1. MHC I = encode HLA - present ____self peptides_______to cell surface
- = present on all nucleated________ cells
- = tells immune system ______________
- 2. MHC II = encode HLA – present ____anitgens___________ to cell surface
- = present on _______antigen presenting cell_________________
- = tells immune system ____something is wrong____________
- 3. MHC III = encode ___blood proteins___________
- = provides non - specific immune function
- Your HLA “type” – transplants, 6 types, 2 in 20,000, must match 4 of 6 for transplant
Human leukocyte antigen genetics
HLA is encoded on Chromosome 6 _________________
Cluster of 200 genes
Encode surface glycoproteins
Importance in:
- 1) Infectious disease
- 2) Transplants
- 3) Autoimmunity
- 4) Cancer
- Antigen processing cells
- APCs
- 1. ____macrophages______
- 2. ___dendrtic cells_______
- 3. ___b - cells_______
- Steps in antigen processing
Phagocytose invader
Digest invader, attach to MHC
3. Move MHC+antigen to cell surface
4. Present MHC+antigen to _____APCs (antigen presenting cells)____
- Immune system overview
- Lines of immune defense
- 1. Physical barriers = skin, mucus, cilia, stomach acid
- 2. Innate System = rapid & non - specific
- 3. Adaptive System = slow & specific & memory
- Mutations can
- 1. ___inhibit________ immune function
- 2. Cause immune ___dysfunction________
- 3. Cause ____autoimmune__________ disorders
- 4. Cause increased infection_______
- 5. Cause ____decreased immune cells_______
- * Mutations can work in positive and negative manners
- immune system - innate
- Rapid, broad defense
- Inflammation = central to this system = creates hostile environment
- Plasma accumulates, area becomes warm, signals immune cells to gather at site
- 3 proteins associated with Innate system (if mutated more susceptible to inf.)
- A.___complement________ = plasma proteins, assist other defenses, MAC punches holes
- B.___collections________ = recognize pathogen pattern sequences, activate phagocytosis
- C.____cytokines_______ = help both innate & adaptive immunity (secreted molecules)
1.___interferon________ alerts immune system to viral infected cells
2.___interleukins________ causes fever = higher body temp = kills bacteria/viruses
3._____tumor necrosis factor______________ = destroys bacterial toxins, attacks cancer cells
- Immune system - adaptive
- Slower, more specific
- 3 characteristics
- 1.__diverse______ = fights many types of pathogens
- 2. specific______ = distinguish between harmful/helpful
- 3. ___rembers________ = reacts faster upon next infection
- 2 types
- 1. ____humoral______ adaptive immunity (B & T cells) = antibody response
- 2. ____cellular______ adaptive immunity (T - cells) = helper & cytotoxic response
- immune system - adaptive - cellular response
- Differs from Humoral Response because T cells travel to where they act rather than _____secerting antibodies_____________ as B cells do
- The life of a T cell
- 1.Comes from ____bone marrow_____________
- 2. Travels to thymus_____ to learn “self vs non self”
- 3. Those that learn survive & go out to the body
- 4. Those that do not learn are destroyed
- 5. Job of T cell is to secrete ____cytokines_______ and ___activate_____ other immune cells
- Types of T - cells
- 1.____helper_____T- cells = recognize foreign antigens, stimulate B cells to make antibodies, secrete cytokines, activate cytotoxic T cells
- 2._____cytotoxic______ T - cells = attack virally infected & cancerous host cells. Touches infected cell, releases perforin to destroy host cell
- 3. ____regulatory_______ T-cells = work to control immune response, avoid autoimmune destruction
- Immune system - adaptive - humoral response
- Antibody response begins when the APC__ activates a T - cell__ which in turn activates a B - cell__
- B cells die in the ___spleen________ and ___lymph nodes_______
- B cells are generated in the ___bone marrow________ & learn self there
- T - cell finds its B cell match it releases cytokines____ that cause the B cell to divide to become:
- 1. ___plasma cell________ = antibody factories (2,000/second)
- 2. memory cell________= become dormant, respond faster
- immune system - antibodies
- Made of several polypeptides = encoded by many genes
- Parts of an antibody
- 1. heavy____ chain = the 2 longer polypeptides
- 2. light____ chain = the 2 shorter polypeptides
- 3. constuant____ region = base of the Y, common to all antibodies
- 4. ____variable____ region = tip of the Y, different among antibodies
- 5. Antigen binding site binds the epitope
- Antibody jobs
- ____inactive______ pathogen, neutralize toxins
- clump_____ pathogens together
- ____activate______ complement system
- immune system - antibodies
- Limitless varieties
- 1. V (variable)
- 2. J (joining)
- 3. D (diversity)
- Heavy chain (chromosome 14) & light chain (chromosome 2 & 22) have 3 gene sections
- Only a few antibody genes, but can make limitless varieties : one for every pathogen you encounter
- Thanks to ______alternative splicing_____________ we can make 10 trillion different antibody combinations
- immune system - antibodies - diseases
- Immunodeficiency with hyper IgM
- 1. Make too much IgM__
- 2. Make too little ___IgG and IgA__________
- 4. Mutation in _____x chromosome____________
- 5. Mutated T cell protein ligand = no signal_____
- Result = can’t maintain long term immunity
- → recurrent bacterial infections
- → risk of autoimmune disease
- Treatment = IV replacement of IgG, bone marrow transplant
- Important immune cells for a healthy system
- Mast cells: Release histamine to recruit immune cells, defend against pathogens, cause allergic reactions
- Macrophages: Phagocytoses cancer cells & pathogens
- Natural Killer: Cells Destroys tumor cells & virally infected cells
- Dendritic cells: Phagocytoses invaders
- Neutrophils: Responds to trauma, kills bacteria & fungi
- Basophils: Releases Histamine, fights parasites, causes allergic reactions
- Eosinophils: Kills bacteria & parasites
- B - cells: Attacks all microbes with antibodies
- T -cells: Stimulates other immune cells & attacks cancer or infected cells directly
- immune system - abnormalities - inherited
- Inherited Immune Deficiencies
- 1. Chronic Granulomatous disease
- →___neutrophils__________ can engulf bacteria, but lack enzyme to destroy
- → Chronic recurrent infections usually bacterial or fungal
- → Enzyme made of 4 different genes = ____all x - linked ___________
- → Treatment: antibiotics & IFN gamma infusion
- → Cure with: Bone marrow transplant or umbilical cord stem cells
- immune system - abnormalities - inherited
- Severe Combined Immune Deficiencies (SCID) Lack of B - cells and/or T cells
- T - cells cannot mature_____ & activate B -cells
- Affects both Humoral & Cellular immunity
- Half of cases are _____x - linked __________
- Die from severe & diverse infections: viral, fungal (thrush), and bacterial infections before age 3
- David Vetter – bubble boy – transplant – Epstein Barr
- immune system - abnormalities - inherited
- Inherited Immune Deficiencies
- 3. Immune defect due to absence of thymus
- No T - cell ___matutation______
- No cytotoxic T - cells for ____viral infections__________
- No helper T - cells to activate ___b-cells_______
- 4. Neutrophil Immuno - deficiency Syndrome
- ___point_____ mutation makes neutrophils defective
- Lots of ____bacterial_______ infections
- Lack of ___pus___ = poor wound healing
- immune system - abnormalities - acquired
- HIV - genome is 1 millionth of ours, has 9 genes – 9,000 RNA bases, encodes 15 proteins – takes over 200 human proteins
- HIV enters__macrophages_________binds to 2 human coreceptors (CD4 & CCR5), once inside reverse transcribes viral RNA into DNA,
- Makes a double helix, enters nucleus, inserts into chromosomes__________
- Cell fills with viral pieces that assemble into new viral particles that bud from the cell. T-helper_____ cells begin to die off
-Bacteria infections begin because B-cells______ cannot activate to produce antibodies.
- Later HIV variants bind to CXCR4 receptors on cytotoxic______ T cells and kill them which allow for cancer and viral infections to take hold.
- immune system - abnormalities - acquired
- Example :HIV
- Steps in infection
- 1. Binds receptor, enters cell
- 2. Reverse Transcription RNA to DNA__
- 3. Make dsDNA
- 4. Incorporates DNA into ___host genome____
- 5. Viral genes transcribed to mRNA
- 6. Viral mRNA to viral proteins
- 7. Capsid surrounds viral genome
- 8. Buds from cell
- How to stop it?
`
- HIV-Treatment
- HIV has no replication repair system so mutates every 5,000 bases.
- Immune system cannot keep up with this ever- changing virus.
- HIV variants arise so quickly that HIV drugs can become useless in days.
- Bone marrow produces 2 billion T & B cells a day, but HIV replicates and buds 1 million to 1 billion new particles a day.
- Combination drugs with different actions offer the best treatment working at different points of infection to slow down HIV = slow lifelong treatable illness.
- Scientists are now looking at people who are resistant to HIV infection. Found variants in 4 receptors or the molecules that bind to them and can block HIV entry. Many had similar homozygous recessive 32 base deletion in CCR5 gene.
- Advantageous mutations
- Chromosome 3
- Heterozygous – delayed__________
- Homozygous recessive –_____no infection__________
- Bone marrow transplant
- Gene therapy
- immune system abnormalities - autoimmunity
- Immune system makes antibodies that attack your own cells
- Polygenic, 5% of the US population, can develop at any age
- Example: Lupus →Autoantibodies attack connective tissue____________
- Example: Ulcerative colitis →Autoantibodies attack ___colon tissue_______
- How does autoimmunity arise
- 1. viral______ infection–host cell proteins
- 2. Immune cells escape thymus____
- 3. Antigen resembles self___- heart valves & Streptococcus
- 4. Skewed ______x-inactivation_______- females have increase in autoimmune
- immune system abnormalities - allergies
- What happens when you have allergies?
- 1. You are exposed for the 1st time to an allergen, but you have no allergic reaction
-B-cells bind the allergen, secrete antibodies (IgE) that bind ___mast____ cells
- 2. You are exposed to the allergen a subsequent time, you have an allergic reaction
-When allergen binds to IgE on Mast cells, it causes them to release ____histamine______ in large amounts
- immune system abnormalities - allergies
- Allergies are an immune response to an allergen___ that is not actually a threat
- Ranges from:
-Hay fever–larger -upper respiratory
-Asthma–small–penetrate lung
-Anaphylactic Shock: IgE binds antigen and causes massive amount of histamine_____ to be released = contraction of bronchial muscles and vasodilation = leaky vessels
- Allergens activate _____T-helper cells______ which in turn release massive amounts of cytokines so both the humor & cellular immunity take part in an allergic response.
- immune system - how to alter it - vaccines
- Smallpox (first one, sort of) 1796 Edward Jenner
- Vaccines = stimulate a__primary______ response to make secondary____ response better (so you are really just tricking your immune system into recognizing it earlier)
- Herd immunity
- 1. Reduces pool
- 2. Limits spread of things
- Smallpox on 3,000 year old mummy
- 1500’s take smallpox scabs grind up to smoke or rub in skin
- immune system - how to alter it - immunotherapy
- Monoclonal antibodies (Mab)
- 1. Recognition– B cells recognize antigen
- 2. production_____– by a B cell of one specific antigen
- 3.___specific______–for only that particular antigen
- Useful for ___diagnosis_______ and ____treatment_________ because they are very specific (pregnancy test– hCG)
- Cytokines
- 1. Interferons = cancer cells, warts
- 2. IL - 2 = administer by IV, treat kidney cancer
- 3. Colony stimulating factor = boost WBC production (AIDs, chemo)
- 4.Tumor necrosis factor = stops binding to self (arthritis)
- mabs & the downside
- ____immune reaction potential_______________ - the antibody type determines the “Immunogenicity potential”
- _____infection potential______________ - Inhibits your ability to fight off infections caused by viruses, fungi, bacteria & TB
- Act only for a short time
- Must be delivered precisely otherwise overdose or side effects
- Will need numerous infusions over time
- immune system - how to alter it - transplants
- Types of transplants
- 1. ___autograft________ = same person (leg vein to coronary artery)*least chance of rejection
- 2. ___isograft________ = identical people (Monozygotic twins)
- 3. ____allograft_______ = non-identical people (kidney)
- 4. ____xenograft_______ = different species (pig heart valve)
- Rejection or acceptance
- 1. It’s a match ____HLA______________
- 2. Drugs _________supress t-cells______________
- 3. Graft v host disease ___transplant attacks_________
- 4. Too good a match ______doesn’t recognize disease________
Genetics of development, behavior, & neurogenetics
- development
Sex is determined upon ______________ (female = X, male = X or Y)
- Oocyte = female germ cell (egg cell)
- Odds are against us all to be here:
- -84/100 oocytes will be fertilized
-of those that are fertilized 68 will be able to implant in the uterus
- of those that implant 42 will survive 1 week
- of those that survive 1 week, 37 will survive 6 weeks
- of those that survive 6 weeks 30 will be born
- The 70 that did not go on ½ have ________chromosome abnormalities__________________so severe development couldnnot proceed
- Within 12 hours of fertilization =
- 1) 2 different nuclei (one mom, one dad) replicate their individual DNA
- 2) 2 nuclei fuse__ = form genetic instructions for a new individual
- 3) Now called a ___zygote______ chromosome abnormalities
- Before birth a female’s million oocytes arrest in prophase I until after puberty. This means when your grandmother was pregnant with your mom the oocyte that would become you was already there.
- Stages of development
- ___germinal_______ Stage
-Fertilization begins this stage
- Covers conception → 2 weeks
- ___embryonic________ Stage
- Embryo & support system forms
- Covers 2 weeks → 2 months
- fetal____ Stage
- All other development occurs
- 2 months → birth
- While protected in the womb still affected by both genetic and environmental factors
- conjoined twins
- 1 in 100,000 pregnancies
- Embryo divides into twins between day 13 and 15
- Are considered identical twins
- Chang and Eng
- Results depend on ____the time point________ that the embryo divides
- Separation surgery very ethically debated
- Chang and Eng (born in Siam which is now known as Thailand 1811 thus Siamese twins). Joined from navel to top of chest. Lived for 63 years, fathered 22 kids and divided each week between their wives (who were sisters). Toured with carnival in the US.
- development
- The nature of birth defects depends on the ___structures that are developing ____________________when the damage occurs
- ____critical period__________ : time when genetic abnormalities, toxic substances, or viruses can alter developing structures
- ___teratogens_______ : chemicals & other agents that cause birth defects
- 1.Thalidomide –limb development
- 2.Cocaine – small growth, language, memory
- 3.Cigarettes – fetal stress & CO inhibits hemoglobin
- 4. Alcohol – intellectual, social, communication
- 5. Nutrients – too much acts as drugs (Vit A & C)
- 6. Occupational hazards – dyes, lead, mercury
- 7.Viruses – can cross placenta (Zika, rubella)
- neural tube defect
- Neural tube
-develops into the ____brain and spinal cord____________
- Neural tube defect = 200 genes identified in mice, not much known in human other than it runs in families
- if neural tube does not completely close by day 28__
- Causes parts of the brain & spinal cord to protrude
- ___Vitamin B & folic acid_____________ added during pregnancy lacking in those with genetic susceptibility
- Blood test during week 15 of pregnancy test for ____alpha fetoprotein__________ in mother’s blood (AFP is released from fetus liver)
- 6 in 10,000 births (2,500 a year) both genetic and environmental causes
- behavior
- Complex response to stimuli that is mediated by both genes & the environment
- The nervous system: Senses the environment → Processes the information → Initiates the response
- ____twin studies________ provide evidence for the role of heredity in behavior response
- Behavior is in response to environmental factors, but how we respond has genetic implications that focus on development, structure, & function of the nervous system and its components
- Behavior is a complex continuum (emotions, mood, intelligence, & personality) difficult to study because:
- 1. There are so many influences such as genetic, environmental, psychological__________
- 2. so many traits that are hard to quantify____
- 3. many behavior disorders are ___wide ranging __________ and have _____overlapping symptoms____________
- To understand behavior, we need to have a little background on two areas:
- Development, particularly ___major time points______________
- Neurogenesis, particularly____how it develops/acts_____________
- the nervous system
- Behavior is an innate_____ & ___learned_____ response to environmental stimuli
- The central_____ nervous system is comprised of the brain & spinal cord
- The peripheral________ nervous system includes nerves outside the CNS, connects them to the limbs
- the nervous system
- Brain is about 3lbs
- 100 billion neurons
- 1 trillion neuroglia _____
- Support & nurture neurons
- Include microglia, astrocytes, & oligodendrocytes
- how genetics plays a role
- 1. Myelin– genes control synthesis of
- Insulates neuron, speeds up signal
- Synthesis starts week 14 fetal development, ends in adolescence
- A. demyelination__________ - loss of myelin sheath signal lost/impaired, nerve withers & dies
- Multiple sclerosis, Guillain Barre syndrome
- B.___dysmyelination _________ - defective myelin, structure from mutation in biosynthesis
- Schizophrenia, Canavan disease, PKU
- how genetics plays a role
- 2. Neurotransmitter synthesis
- A. Too little = depression_______ - lower than normal levels of serotonin______
- Medications block reuptake of serotonin so more stays in the synapse
- B. Too much = ___serotoin syndrome______________: Hypomania, Hallucinations, and Tachycardia
- how genetics plays a role
- Neurotransmitter synthesis
- A. Too little =
- depression________- lower than normal levels of serotonin
- Medications block reuptake of serotonin so more stays in the synapse
- how genetics plays a role
- 2. Neurotransmitter synthesis
- B. Too much = ___serotin syndrome ______________- excessive levels of serotonin that overstimulates the post synaptic neuron
- Causes hypomania, hallucinations, tachycardia, tremors
- how genetics plays a role
- 3. Reuptake transporter protein
- A. Can become blocked______ =
- Cocaine block dopamine reuptake
- Plays a role in addiction
- B. Can become ___mutated______ =
- A missense mutation causes Panic disorder
- SNP here is linked to ADHD
- how genetics plays a role
- 4. Receptor + neurotransmitter binding
- A. Blocked =
- B. Mutated =
- Both interrupt signals as in Myasthenia gravis
- antibodies_______ block signal between neurotransmitter acetylcholine & receptors at neuromuscular junctions
- Inhibits excitatory effect of acetylcholine
- Muscle weakness involving eye, facial, talking, chewing, swallowing
- epigenetics and development
- You have external experiences (stress, lack of nutrition, toxins)
- Neural signals start production of gene regulatory proteins in cells
- Proteins add/remove epigenetic markers
- Epigenetic markers control if a gene is turned “on” or “off” during brain and human development
- behavior disorders, genetics & synapses
- Huntington disease (HD)
- Single dominant defective gene on Chromosome 4
- If you inherit the gene you will develop HD ( Autosomal Dominant – ___complete________ penetrance)
- Huntington protein function unknown
- Brain changes: Uncontrolled movement, decline in thinking & mood (depression, anxiety, anger)
- Typically develop between age 30- 40
- ___CAG_____ repeats = less than 35: normal, more than 36: HD
- MRI findings = lateral ventricle atrophy (cells have died, filled with fluid)
- behavior disorders, genetics & synapses
- Alzheimer disease - Most common cause of ___dementia_________
- Early memory deficits → gradual cognitive & intellectual function decline
- Characterized by
- 1. Loss of neurons & synapses in hippocampus, cerebral cortex, subcortical region
- 2. Buildup of ______plaques & tangles__________
-6th leading cause of death in the US
- Live avg of 8 years after symptoms develop
- No cure, current treatment slows symptoms, extends life
- Eating disorders
- 5-10 million people in the US have eating disorders
- Includes anorexia nervosa & bulimia, but also other forms such as muscle dysmorphia
- Considerable genetic component : - 50-70%_____ is genetic, rest environmental
- 0.8 heritability, Chromosome 1 & 10
- Environmental: western society: thinness & beauty, higher rates of eating disorders.
Genetics for increase in hunger
- Leptin – tells hypothalamus to ___decrease______ appetite
- Leptin transporter – moves leptin from blood to brain__
- Leptin receptor – binds leptin to hypothalamus
- Melanocortin receptor – activates when leptin is high_____
- PYY_relays signal from stomach to brain when full
Genetics for increases in hunger
- neuropeptin Y____- made when leptin low to increase hunger
- Ghrelin____- stimulates neuropeptide Y
- Stearoyl COA____ - controls the use or storage of fat
Sleep
- Vital behavior, unknown function (spend 1/3___ of our lives sleeping)
- Sleep genetically controlled, environment impacts duration & intensity
- Narcolepsy________ = polygenic trait with environmental trigger
- Occurs in 0.06% of population but common in families
- receptor for neuropeptide does not reach cell surface – brain cannot get signal to stay awake
- Sleep paralysis = can’t move after waking & Cataplexy
= fall asleep after laughing/excitement
- Familial Advanced Sleep Phase Syndrome = circadian pacemaker, light/dark cycles
- Utah family: single base pair mutation: asleep at 7:30pm wake at 4:30am (autosomal Dom)
- Period2 mutation: 2 adenines = early riser, 2 guanines = late sleeper, heterozygotes in between
- Neurotransmitters
- Sleep promoters: adenosine (coffee counteracts), serotonin
- Awakeness promoters: histamine, dopamine, acetylcholine
- Using Genome Wide Study found the ABCC9 gene that if you have a different SNP than the rest of the population you only need ½ the amount of sleep
Intelligence
- Intelligence is the use of mental skills to complete tasks or solve problems
- 3 in 100 have intellectual disability in US
- Use a “g” IQ scale to test for generalized intelligence & not enhanced opportunities
- Average score 100
- Score 50-70 = mild intellectual disability
- Score <50 = severe intellectual disability
- Environmental or Genetic? Heritability changes over time
- chromosomal disorders have intellectual
- Disability environment: poverty, divorce rate, incarceration
- As a person ages they have more control over their environment so genetic contributions become more prominent
Drug addiction
- Two identifying characteristics of drug addiction are:
- 1. tolerance________- need to take more to achieve the same effect as time goes on
- 2. dependence _________ - onset of withdrawal symptoms upon stopping use of drug
- Combination of genetic , biological, and psychological components of craving the drug heritability is between 0.4-0.6
- Receptor that binds acetylcholine binds cigarette nicotine →stimulates dopamine release. This receptor is also found on lung cells where it binds carcinogens
- gene variant increases desire to smoke after first cigarette
- Nicotine causes addiction, susceptibility to lung cancer, delivers carcinogen
Drug addiction
- Genes implicated in addiction are those that have protein products that are Part of postsynaptic transduction pathway: neurotransmitter production, reuptake transporters, receptors on postsynaptic neurons
- THC, cocaine, opium & other plant derived drugs bind receptors on human neurons = this means that our bodies have versions of these plant derived compounds
- Opiates (heroin & oxycontin) equivalent to ___endorphins________ (dull perception of pain & elevate mood)
- THC equivalent to ____anadamide___________ (response to stress & social behavior)
- LSD equivalent to trace amines
- The A1 allele of dopamine receptor gene DRD2 is more common in people addicted to alcohol & cocaine = however your genetic disposition will not doom you to a life of addiction
Mood disorders - depression
- 6% of general population at any time (women more than men)
- Genes & environment contribute equally__________
- Genetic component remains unknown (if a family member has depression you are 5x more
likely to have depression) environment: pollution, alcohol, stress, trauma, toxants
- Serotonin, norepinephrine deficiency
- Selective serotonin reuptake inhibitors allow more serotonin to stay the synapse
Schizophrenia
- Debilitating loss of ability to organize thoughts, perceptions, form of psychosis, delusions, hallucinations, voices giving instructions
- Genetic influences: Rare duplications, deletions Heritability 0.8____ Identical twin has it, you have 50% chance
- Environmental influences: Birth complications, Fetal oxygen deprivation, Viral infection at birth, Malnutrition, Mom – brain injury Living in cities, migration
Age begins = Males (17 - 27) Females (20 - 37)
Autism Spectrum Disorder
- Loss of communication and social skills (around 3 yo) & affects 1 out of 68 children (CDC 2014)
-5x higher in boys & 90% heritability
- 30 susceptibility or causative genes
- Proteins (gene products) and environment?
- Neurexins & Neuroligins are found in the pre & post synaptic neurons.
- Mutation causes misfolding, impairs signal
- 2 sets of synapses form during development:
1)those before birth___, 2) those in early childhood in response to ______experiances_________
Genetics of aging, longevity, & late onset disease
Maturing and aging
- Aging (definition) = the process of becoming older, in humans, aging represents the accumulation of changes in a human being over time
- Based on 3 perceptions
- 1.____chronological age___________ = based solely on the passage of time. Age in years. Limited significance in terms of health
- 2. ___biological age____________ = based on changes in the body (particularly physiological) that occur as people age
- 3. _____psychological age__________ = based on how people act and feel
Normal aging (AKA getting old sucks)
- cells____ – as cells age, function less. Genes control programmed cell death. Cells can only divide a limited number of times (telomeres)
- ___organs_____ – made of cells, when # of cells becomes too low, organ cannot function normally
- ____bones and joints___________– bones become less dense, weaker, prone to breaking, cartilage breaks down, ligament less elastic
- ____Muscles & Fat__________ – muscle mass & muscle strength decrease around age 30, lose fast twitch fibers, lose 15% muscle over adulthood. Body fat doubles by age 75.
- Eyes____ – lens stiffens (impairs focusing, after 40yo trouble with objects 2ft away), becomes dense (impairs light- 60yo needs 3x the light), yellows ( impairs color), reduced nerve cells (impairs depth perception), less fluid (feel dry)
- ears_– hard to hear high pitch sound, harder to understand letters (sound mumbled), more ear wax (ewwwww)
- ___mouth & nose____________– 50yo taste and smell diminish, nose gets bigger, dry mouth, taste buds less sensitive
- skin_____ – less collagen so tears easier, less neve endings, thinner, wrinkling, drier. Melanocytes decrease, brown spots
- ___Brain & nervous_____________ -# of nerve cells decreases, blood flow decreases, reaction and time to perform tasks slows, signal conduction decreases
- heart & blood_____________ – become stiffer, blood pressure increases, during exercise the heart cannot speed up as quickly or pump as fast
- lungs______ – muscles weaken, number of alveoli decreases, less oxygen absorbed,
less able to fight infection
- ___digestive_________ - less affected than other body parts, food moves through slower, can’t hold as much
- Kidneys_____ – becomes smaller, at 30yo filter blood less efficiently
- ____blood____ – bone marrow becomes less active , less blood to when needed
- ___immune system_____________ – immune cells act slowly, vaccines less effective, more infections, more cancer, less allergies
Adult-onset inherited disorders
- Genetic diseases that do not present until ___adulthood_________
- Due to
- 1)___inherited_______– Accumulation of gene products, Accumulation from birth gradually over decades, Genetic malfunction of substance that normally break them down
Example: Huntington disease
- 2)____acquired______ - Accumulation of harmful mutation, Some present at birth, most from exposure to mutagens, Mutations that activate dormant genes.
Example: Cancer
Adult-onset age related diseases
- Cancer
- Longer we live, more mutations__more mutations________ we accumulate
- We are living longer = increased cancer rates
- If you live long enough you will develop cancer
- Men: 1 in 2 risk developing (prostate cancer #1)
- Women: 1 in 3 risk developing (breast cancer #1)
- 10%___ of cancers are genetically inherited
Adult-onset age related diseases
- Diabetes
- 1 in 4 over 60 will develop diabetes
-Particularly Type 2 diabetes
- Insulin regulates glucose
- Cells resistant to insulin → little insulin → sugar builds up in bloodstream
- 73%_of developing diabetes is genetic, the remaining is contributed to age, race, obesity, inactivity, viral infections
- Type I diabetes has been associated with variants of the HLA genes__________ ( immune recognition self v non-self)
Adult-onset age related diseases
•COPD
- Chronic inflammation of airway = obstruction
- Emphysema & bronchitis
- 1% is genetic, the rest is environmental (smoking, particulate exposure)
Adult-onset age related diseases
Macular degeneration
- Leading cause of vision loss over age 50
- Macula____ breaks down = blurred vision in center
- Women 64% more likely develop AMD than men
- Genetics account for ____ age & environment account for rest
- 1 in 500 chance for 55-64 year olds
- Increases to 1 in 8 for 85+ year olds
- Those who work out 70% less likely to develop
- 89% of those with AMD are caucasian
Adult-onset age related diseases
- Alzheimer’s
- NOT a normal part of aging
- Early - onset (before age 60) - 3 genes
- 100% genetic
- Late - onset (after age 60) - not identified
- Early - onset almost 100%_____ genetic
Adult - onset age related diseases
- Immune dysfunction (Rheumatoid arthritis)
- Immune system attacks membrane in joints, pain, joint malformation
- not___ hereditary, genetics that affect immune function contribute
- Mutations in ___ HLA genes_______
- Environmental influences include hormones, occupational exposure to fibers, microbial infections, long term smoking & age
- underlying factor of all accelerated aging diseases = genomic instability
Hutchinson Gilford Syndrome
- Progressive disorder causes children to age rapidly (8x faster)
- Develop normally until 9 months – 2 years
- Average lifespan 13 years
- Lamin
- A = typically makes a protein for holding the nucleus together
- Mutation of Lamin A abnormal protein made = progerin ________
- Children develop aging diseases = arthritis, cataracts, cancer, hair loss, strokes
- Typically die of complication related to atherosclerosis
Werner syndrome
- “adult progeria” = develop normally until puberty
- Advanced aging around in 20’s
- Develop age related disorders = cataracts, type -2 diabetes, atherosclerosis, osteoporosis, cancer
- WRN gene = maintenance & repair of ____DNA ______
- Mutation = short nonfunctional protein, doesn’t reach nucleus to repair DNA
- Cells slow or stop dividing
Genes and longevity
- Average age of life expectancy
1960 2018
69.7_____U.S. 79.9____
___66.6____Men___76.6_____
___73.1____Women__81.4___
- Oldest living person ever: 122 years 164 days (Jeanne Calment, France, 1875 - 1997)
- Centenarians (100 years old): 1 in 6,000
- Supercentenarians (over 110 years old): 1 in 7 million
- On average people are living longer, but those passing the upper limits have dramatically plateaued.
- Genetic and environmental components
Genes and longevity - genetics
- Look at Centenarians (those that live to 100 ) and Supercentenarians (those that live to 110+)
- Mice = 2011, breast cancer drug ___tamoxifen__________ – reversed the symptoms of aging
- DNA damage & Telomeres
- Twin studies – genetics accounts for 20 - 30% of an individual’s chance of surviving to age 85
- Study found that centenarians have increase in
- 1) gene variants that confer ____disease reistance____________
- 2) gene variants that _____counteract_______ effects of disease associated genes ____chromosome II________– region shared among siblings that live to be 90+
Genes and longevity - genetics
- Single genes important for aging
- Insulin control
- Glucose metabolism
- Immune System function
- Cell cycle control
- Cholesterol metabolism
- Stress response
- Antioxidant production
- APOB & APOE = protein involved in metabolism of lipids
- ACE = controls blood pressure by regulating volume of fluids in the body
- HLA-DR = MHC class II cell surface receptor on T- cells
- SOD2 = enzyme that breaks down mitochondrial by - products
- TH = enzyme that helps make dopamine and norepinephrine and epinephrine Cytochrome P- 450 = metabolizes thousands of chemicals, found in mitochondria and ER
Environment and longevity
- ____diet, smoking, exercise________________ – determines how long & how well you age
- ____7th day adventist ___________ –encourage behavior that promotes healthy aging, live 8 years longer
than average US citizen (don’t smoke or drink alcohol, exercise regularly, vegetarian)
- ____calorie restriction___________
- 30% reduction in the amount of calories consumed
- Live 40% longer
- More resistant to age related diseases
- antioxidants_______= Reducing agents, limit oxidative damage by neutralizing free radicals Examples: Vitamin A, C, E, beta - carotene, dark chocolate, red wine
Telomere aging
- Shorten as we age
- Once they reach _____critical length________, cells either:
-Die off
- Enter ___senescence_________ = cells cease to divide - Human fibroblasts (50x)
- Hyper long telomeres in mice
- Kept the long length over time
- Less DNA damage___
- Better DNA repair___
- Fewer tumors ___
Oxidative stress
- Oxidative stress = imbalance between _______pro-oxidants & antioxidants________________
- ____free radicals________theory of aging = we age because cells accumulate free radical damage over time
- Free radicals = any molecule that has a single unpaird________ electron in outer shell
- Examples
- 1. Superoxide O2-
-2. Hydrogen peroxide (H2O2)
- 3. Peroxynitrite (OONO)
- Comes from = poor diet, smoking, alcohol, lifestyle, sun, too much exercise, too little exercise, fast food, air pollution, medication, pesticides.
- Just about everything we do results in producing free radicals
Glycation
- Glycation = _____non-enzymatic rxn_____________between reducing sugars and proteins/lipids/nucleic acids (macromolecules)
- Stiffness and loss of elasticity in arteries & organs
- Reduction of sugars causes accumulation of glycation end products in the extracellular matrix
- Protein molecules then crosslink sugar molecules = advanced glycation end products (AGEs)
- Body does not recognize as normal, makes antibodies to attack = ____inflammation__________
- Deposits into skin = ____wrinkling ______
Genetic technology: pcr & sequencing
PCR - what goes in
- Mimics what happens in your cells when they undergo DNA replication
- primers____ known sequence flanking area of interest
- ______DNA polymerase_________ enzyme required to add nucleotides to new strand
- template DNA________ what you want to amplify from
- ___nuceotides_________ to add on to the amplified strand
PCR thermocycler steps
- 1. Initial denaturation
- 2. Denaturation
- 3. Annealing
- 4. Extension
- 5. Final extension
- denaturation, annealing, and extension are repeated 30-40 cycles
PCR thermocycler steps
1. Denaturation
- Typically, performed at 94°C
- Allows for full denaturation of the template strands, especially long DNA strands (breaks ____hydrogen bonds________ between strands)
- Will be repeated each of the 30 - 40 cycles
PCR thermocycler steps
- 2. Annealing
- Typically, at 54°C
- Lower temperature allows primers to anneal__ to template
- Have 2 primers (forward & reverse) for each of the parent strands
- Will be repeated each of the 30-40 cycles
PCR thermocycler steps
- 3. Extension
- Typically, at 72°C
- Optimal for DNA polymerase to bind to primer/template
- Adds free nucleotides______ to new strand
- Will be repeated each of the 30-40 cycles
PCR thermocycler steps
- 5. Final Extension
- Typically at 72°C
- Makes sure all strands are completely finished
How to see your PCR
- Gel electrophoresis
- DNA is negatively ________charged
- If you apply an electrical current it will migrate (from = – to +)
- Use a chemical like ethidium bromide to visualize under UV
First Generation sequencing
- Sanger Sequencing (Aka.....chain termination method or dideoxynucleotide method)
- Uses only one strand of the double stranded DNA
- Start synthesizes new DNA strand using primer
- Uses dideoxynucleotides (ddNTPs = ddG, ddA, ddC, ddT) & regular nucleotides
- When a ddNTP is incorporated, ___elognation stops___________
- Results in numerous fragments with ddNTP on end
- Separate fragments on gel, align sequence and read it
Sanger sequencing
- How to read a Sanger Sequence
- Start at the top and work down the gel
- 1st use = 1977
- Used for decades, some still today
- Drawback = ___cost & time__________
Second generation sequencing
- Called “sequence by synthesis”
- Benefits over 1st Generation Sequencing:
- Millions of short reads in parallel
- Faster & low cost
- Don’t have to do gel electrophoresis
Second generation sequencing
- Fragment DNA
- Run PCR to add adenosine_________ to ends that primers attach to
- Add to flow cell that has adapters
- Primers bind to adapters
- Amplified and bends to match next adapter
- Fluorescently labeled nucleotides bind during amplification
- This gives a color signal to record the base
Third generation sequencing
- Called “single molecule real time read”
- Benefits over 1st Generation Sequencing
- 2 nd Generation requires an initial round of PCR first, 3rd Generation does not
- Complex genomes with lots of repeats confuse 2nd Generation, 3rd Generation does not
- 3rd Generation does not need chemical labeling
Third generation sequencing
- Nanopore
- _____single strand _____________DNA is fed thru a small pore opening (10-9 meters)
- Constant electrical field is generated
-Each nucleotide changes the current differently
Sequencing coming to a bedside near you
- Illumina $30,000 sequencer = human genome 1 hour = $1000
- Establish in hospitals to:
- Avoid cost of drug/therapy that has little chance of success
- Avoid/limit patient side effects
- Decrease disease burden looking for right treatment
- Act in preventative manner by predicting susceptibility
- Making correct diagnosis
- Monitoring and typing infections
Genomics - human genome project
- Goals (started 1990 by Watson, used WBC 2 males & 2 females, started 1990 & finished 2003) Publicly funded
- 1. ____identify_____ all human genes
- 2. ___map location_________ of all human genes
- 3. ____sequence________ all 24 chromosomes (looked at only euchromatin)
- 4. Analyze ___variation_______ between humans (ex. SNPs, VNTR)
- 5. Map & sequence genomes of _____model organisms _________ for future medical research
- 6. Develop new _____genetic technologies______________ to speed up research
- 7. Give findings to other scientists & ___general public__________
- 8. Set up ___ELSI__ program (Ethical -Legal- Social)
Genomics - human genome project
- How to sequence a genome
- Step 1 : Cut the genome into ______small overlapping pieces_________________
- Step 2 : Determine the ____sequence________ of smaller pieces
- Step 3 : Use ____algorithms________ to find overlaps
- Step 4 : overlap________ pieces to determine sequences
- * However, using this method missed CNV__
Sanger Sequencing
- Frederick Sanger, 1977
- Basically combines PCR___of unknown DNA segments and ___gel electropheresis_____________
- Uses _____fluorescently___labeled__________ dideoxynucleotides (lacks a ____3’ hydroxyl group ________)
Genomics - human genome project
- Gov’t funded:
- _____clone by clone method__________________
- _____1 chromosome @ a time__________________
- ________slower_______________
- ________doesn’t miss repeats_______________
Celera Genomics (Craig Venter)
- ___whole genome shotgun____________________
-_____entire genome at once_________________
- ______faster_________________
- _______misses repeats_______________
Genomics - human genome project
- The results:
- HG = 3.1 billion nucleotides
- Less than 2% of the HG is protein coding
- We are all 99.9% similar (SNP & CNV)
- Average size of a gene = 25kb
- 50% sequence similarity to other organisms
- Genes not uniformly distributed (Chromosome 1 most, Y chromosome least)
- Largest protein gene = Dystrophin (2.5Mb) (1Mb = 1,000,000 bp )
Genomics - human genome project
- Cooler ways to sequence came from the HGP $3 billion & 10 years → $1000 & few hours
- Chain termination - Fluorescent labeling
- Nanomaterial
Genomics - proteomics
- Proteomics is the identification, characterization, & quantitative analysis of the proteome of a cell, tissue or organism
- Which is larger: proteome or genome?
- Due to protein ____modification__________ & ______alternative splicing _____________
- Looks at :
- 1. Protein - protein interactions
- 2. Protein regulation
- 3. Protein modification
- 4. Protein location
- 5. Protein detection & quantization
- Methods
- 1. Isolate proteins from a _____single organelle__________________
- 2. Isolate entire protcome__________ Three areas of proteome analysis
- 1. ___expression__________ proteomics
- 2. ____bioinformatics___________ proteomics
- 3. ____functional___________ proteomics
Genomics - microbiome
- Human Microbiome Project 2007 - 2012 ($115 million)
- Sequenced the bacterial___, fungi__, & viruses__ that make up the microbiome of 250 healthy adults
- Sampled 17 areas on body and sequenced the 16s rRNA________ of bacteria
- Goals
- 1. Do humans share a core________ human microbiome?-
- 2. Does the microbiome ____change with health____________________
- 3. _____develop new technology__________________ to analyze the microbiome
- 4. Address ___ELSI____________
Genomics - microbiome
- Obtained 1000x more sequence data than the HGP
- 10,000 different bacterial species, 100 trillion
- Microbiome starts at birth
- We all have different microbiomes with similar groups
- More diverse = healthy
- Less diverse = unhealthy
- Smoking v pregnancy
- IBD & C. diff
- Twins
- Microbe fingerprint
Microbiome - brain/gut axis
- Brain - gut axis
- 1.__lymphocytes___________ sense gut lumen, release cytokines
- 2. _____vagus nerve________ terminals activated by gut microbial peptides
- 3. ____neurotransmitters_______________ made in response to microbial metabolites
- 4. hormone______ release via hypothalamic pituitary, modulates gut composition
- 5.__neural activation______________– sympathetic activation produce neurotransmitters modify microbiota composition
- How do we study the BGA?
- 1. ___germ-free mice_____________ = mice born devoid of any normal bacterial flora
- 2. ___probiotic_____________ = Lactobacillus & Bifidobacterium reduce anxiety levels, lower inflammatory cytokines, reduced depression
- 3. _____antibiotic intervention___________________ = reduce biodiversity, delays re - colonization
- by probiotics, expression of GI symptoms
- 4. infection____ = increases inflammation, anxiety, Campylobacter pathogen activated brain regions associated with processing GI sensory information
Genomics - metagenomics
- Metagenomics is also called ___environmental genomics______________ and uses the ____whole genome shotgun____________approach to sequence the genomes of entire communities of microbes in environmental samples of water, air, soil.
- Want to understand
- 1. ___interactions________ of microbial communities & environment
- 2. ___classify________ new species
- 3. Identify genes with novel functions__________ (medicine/biotech)
- 4. Be able to isolate DNA directly from ____sampled area___________
- The Sorcerer II Global Sampling Expedition (Craig Venter)
Genomics - other “omics”
- ___metabolomics__________ : Study of proteins & enzymatic pathways in cell metabolism
- gkycomics__________ : Study of carbohydrates of cells or tissue
- ____toxicogenomics__________: Study of the effects of toxic chemicals on genes
- ___transcribtomics___________ : Study of all gene transcripts in tissue or cells
- _____stone age genomics____________: Study of ancient DNA (oldest 70,000)
- ___nutrigenomics______________ : Study of the interactions of diet and genes
Genomics - systems biology
- Is an intergrative__________ process
- Interprets ____genomic information_________________ in the context of _____biological pathways___________
- ____interactome___________ is the
- 1. Protein to protein interactions
- 2. Protein to nucleic acid interactions
- 3. Protein to metabolite interactions
- ___network map___________ – shows interacting proteins, genes, & molecules, but cannot tell you when & where the interaction occurs
- Important for:
- 1. Scientists – model potential interactions in normal and disease processes
- 2. Pharmaceuticals – drug discovery, development, detection
Genomics - bioinformatics
- Bioinformatics = the use of _____computers & mathematics _______________to organize, share, and analyze data related to gene and protein sequence____, structure, function & expression
- ____databases_______ are essential for the storage and sharing of all this newfound information
- gen bank____ is the largest publicly available database of DNA sequences (doubles in size every 14 months, 100,000 different species)
- BLAST will tell you
- 1. Genes with similar sequences
- 2. E - values = matching the sequence based on chance
- 3. Identities = similarity score based on alignment
- 4. Gaps = deletions or insertion points
Pharmacogenetics & personalized medicine
The ACCE test
- A = ___analytical_______________= how well does the test measure what it claims to measure (accuracy)
- C = ___clinical valibdation_____________= how well does the test predict the health outcome that it claims
- C = _____clinical utility__________= how useful are the results to the patient
- E = ____ethical aspects___________ = is it voluntary, insurance, employment
Pharmacy meets genomics
- First step to personalized medicine = personalized prescribing
- Medicine has different effects on different patients
- Some patients have different sensitivities_______ = higher/lower dose
- Some individual patients have no__ therapeutic effect
- Some individual patients have ___adverse____ reactions
- Some patients cannot take a drug due to ____interactions________ with another drug
MTHFR genetics
- Considerable evidence of folic acid and diseases like depression, bipolar disorder
- MTHFR gene mutations = can’t convert folate to biologically active form Methylfolate
- Methylfolate essential for making ___neurotransmitters___________ serotonin & dopamine
- This can both cause depression AND cause you to be resistant to antidepressive treatments
- No Methylfolate = cannot add methyl___ groups
- Due to SNP in one of two areas of MTHFR gene (50% of population has at least 1 SNP)
- The more SNPs mean the greater it :
- Reduces response to antidepressants
- Reduces response to chemotherapy
- Increases adverse reactions to nitrous oxide (dental anesthesia)
Terms
- pharmogenetics_____________ = study of the roles of specific genes in drug interactions
- ___charmogenetics______________ = uses genome - wide studies to study drug interactions
- ___pharmokinetics_______________ = studies absorption, activation, metabolism, excretion of drugs ( what the body does to the drug)
- ____pharmodynamics______________ = studies the actual target response (what the drug does to the body)
How genetics play a role in medical drugs
- Genetic factors are all part of the different pharmacy + genetics field
- 1) ___absorption_________ = differences in how patients differ in ability to transport an oral drug to
bloodstream
- 2) ___acrivation_________ = drugs that are given in the form of a prodrug that have to undergo
enzymatic reactions (by the liver) to become active
- 3)____target response____________= differences in how patients process or pathway that is targeted by
the drug response due to local concentration
- 4) _____catobolism and secretion_________________= differences in how patients break down & dispose of the
drug (slow metabolizers have longer/stronger response to drug that fast metabolizers)
- Adverse drug reaction in US = 100,000 deaths/year
- Wasted time in treatment, wasted money, extra suffering
How drugs are metabolized & genetics
- Phase 1 reactions = produce the ____biologically active_______________ molecule
- Polymorphic variations in enzymes among people alter these reactions
- CYP2D6 enzyme= used in metabolism of 25% of all drugs
- poor, intermediate, extensive, ultra rapid metabolizers
- poor = codeine is ineffective______
- ultra rapid = codeine has ____increased risk_________of sedation/impaired breathing
- also linked to beta blockers for hypertension & antidepressants
How drugs are metabolized & genetics
- Phase 2 reactions = produce water soluble molecule for ___excertion________
- Polymorphic variations in enzymes among people alter these reactions
- Thiopurine methyltransferase (TPMT):
-transfers methyl group to immunosuppressant drug azathioprine
- ___heterozygotes___________ = normal elimination of the drug
- ___homozygotes___________ = life threatening bone marrow toxicity due to poor elimination
Where pharmaceutical companies come in
- Want to make $ by selling drug to widest possible range of patients: Pharmaceutical genetic medicine: 15 years, $1 billion
- Merk 2004 Vioxx anti-inflammatory for arthritis increased heart attack/stroke
- Stages of development:
- ___preclinical__________= lab studies in vitro & animal = explores toxicity & pharmacokinetics
- _____microdosing__________ = single small dose given to 10 - 15 healthy volunteers (first to involve people)
- explores preliminary pharmacodynamics & pharmacokinetics
- Phase 1 = 100 healthy volunteers
- explores general safety, tolerability, goal how to _____deliver_____ (pill, injection, etc ) & what is the highest dose
- Phase 2 = 100’s of patients
- explores general safety, tolerability , survival time, _____quaility of life_________
- Phase 3 = 1000’s multi - centers & Worldwide, randomized 10,000+ patients Assigned to 2 groups: 1) current treatment vs 2) new treatment ___double blind__________ so participant & research team don’t know who gets what treatment Last step before ___FDA approval_______
- Phase 4 = Post market _____surveillance___________ (largest group: 100,000+ patients)
- explores adverse reactions, long term health implications & off label uses
Genetic medicine - treatment - pharmacogenetics
- Typically work in 1 of 3 ways
- 1. Enzyme ___replacment_________ therapy = recombinant human enzyme infused to
- compensate for deficient enzyme (LSD – no enzyme to breakdown, must infuse, $500,000 year)
- 2. Substrate reduction_______therapy = oral drug that reduces the level of substrate so that the enzyme works more efficiently
- 3. Pharmacological ___chaperone_________therapy = oral drug that binds misfolded protein so that the protein function is restored
- Lysosomal storage disease example (Gaucher disease)
- Antibiotics (aminoglycosides) – works on bacteria by shutting down ____protein synthesis__________ , but also affect nonsense mutation____________ in humans by distorting the ribosome so that protein synthesis to continue. Currently, about 12%_ of all mutations are nonsense
A closer look at cystic fibrosis & treatment based on genotype
- CFTR gene
- 2000 known mutation
- F508___deletion
- 90% of CF patients (27,000)
- October 2019 FDA approved Trikafta
Expression profile of tumors & treatment
- Traditional approach classify stage________:
- tumor size
- involvement of lymph nodes
- presence of metastases
- Personalized medicine approach _____gene expression_________:
- Microarray to analyze tumor RNA
- Identify genes that are up/downregulated
- + stage cancer (early vs late)
- + good or poor survival outcome
- + treatment tailoring
Success of personalized medicine: biomarkers
- predisposition __________ biomarkers = genetic susceptibility.
- How likely is a person is going to develop a health disorder if they have a certain biomarker.
- Example: BRCA1&2 indicates an increased susceptibility to breast cancer (family history)
- Guides persons future medical care
- diagnostic________ Biomarkers = genetic confirmation.
- Does a patient have a certain disorder.
- Example: CFTR. Presence of mutation would indicate someone has cystic fibrosis.
- Earlier detection than physical examination or symptoms
- prognostic_______ Biomarkers = Genetic progression.
- How a disease may develop in an individual when they have been diagnosed.
- Oncotyping looks at 21 genes to determine likelihood breast cancer will come after initial treatment
- ___predicitve _______ Biomarkers = Genetic treatment.
- Helps to determine what treatments will work best in a particular patient with the least side effects
Other examples of personalized medicine - the need to screen for genetics
- HIV – Abacavir is a highly effective treatment for HIV, but 8% of patients suffer severe side effects.
- HLA - B 5701 gene variant causes ___hypersensitivity______________to the drug
- Can now screen patients for the variant & give an alternate treatment
- Rheumatoid arthritis – Azathioprine is an immunosuppressant used
- Some patients can’t break down drug, builds up in bone marrow killing immune cells____
- Variant of TPMT gene causes this, can now screen patients for the variant
- Pulmonary embolisms – Warfarin given to reduce blood clots______
- Must give at correct dose = too little no effect (blood clots), too much excess bleeding
- VKORC1 gene variation affects a patient’s sensitivity to Warfarin
Hurdles to personalized medicine
- Regulatory oversite – FDA So far there is a lack of standards that have not been addressed
- Intellectual property rights US Supreme Court – naturally occurring genes cannot be patented
- Reimbursement How & when to be charged, who pays
- Privacy & confidentiality
- Availability of tests Right now, so many conditions do not have an approved test
- No current bedside test
Gene therapy
- (def.) introducing cloned genes into living cells to treat disease
- 7000(+) human genetic disorders due to single gene abnormality
- First gene therapy trial in 1989
- 1,700 approved clinical trials since then
- Currently only 2 gene therapies approved by the FDA
- 1) Aug 30, 2017 = Kymriah for acute lymphoblastic leukemia
- 2) Oct 18, 2017 = Yescarta for non-Hodgkin lymphoma
First ever gene therapy
- 1989 – patient with SCID
- Adenosine Deaminase Deficiency
- AD enzyme needed in purine metabolism
- Worked, but needs continuous treatments
- 2000 –X-linked SCID, 5 of 10 developed leukemia & died
- 2001 – Liver enzyme deficiency, massive inflammatory response, multiple organ failure, death
Candidates for gene therapy
- Criteria:
- 1) Disease causing gene identified ______
- 2) Can clone____/synthesize gene in lab
- 3) Cells for treatment are _______accessible____
- Is the ultimate goal of Translational medicine in a “bench to bedside manner”
- All gene therapy is somatic____ gene therapy
- Currently there is not ____germline____ gene therapy
Approaches for gene delivery
• Two forms:
1) In-vivo
- Gene delivered ___into patient________
2) Ex-vivo
- Cells taken ____out of patient_________
How to deliver genes
- 1. Viral vectors
- (A) Retroviruses
- (B) Adenoviruses
- (C) Lentiviruses
- 2. Non-viral vectors
- (A) Liposomes
- (B) Nanoparticles
- (C) Gene pills
- (D) RNAi
How to deliver genes - viral vectors
- (A) Retroviruses – has an RNA genome
- Process:
- Harmful genes of virus (replication & disease causing) removed
- Cloned human gene inserted into retrovirus
- Infect patient cells
- RNA made into DNA (reverse transcription), inserts into host genome
- Advantages
- (+) = insert into host genome so it is stable__
- (+) = have long term_____ gene expression of correct protein
- (+) = when host cell divides the correct gene is in all new cells______
- Disadvantages
- (-) = can only infect dividing_____ cells
- (-) = can cause toxicity_____ by insertional mutation
- (-) = can ___inactivate_______ essential genes by inserting into them
- (-) = can insert into promoter region causing cancer_____
How to deliver genes - viral vectors
- (B) Adenoviruses
- Process:
- Cloned human gene inserted into adenovirus
- Infect patient cells
- virus delivers genes to nucleus that make an episome_____
- Advantages
- (+) = Can carry medium____ sized genes
- (+) = ___does not_______ inactivate essential genes or cause cancer
- Disadvantages
- (-) = can only infect dividing cells
- (-) = If you have ever been infected by adenovirus immune system will attack_______
- (-) = ____does not______ integrate into host genome so treatment is only ___temporary________
- (-) = when host cell divides, episome is not__ in all new cells
- (-) = patient will need continuous new gene therapy treatments
How to deliver genes - viral vectors
- (C) Lentivirus – a retrovirus (includes HIV)
- Process:
- The disease causing portions of HIV are removed
- Correct gene is inserted into lentivirus
- Infect patient cells
- RNA made into DNA (reverse transcription), inserts into host genome
- Advantages
(+) = Can carry large___ genes
(+) = can__ incorporate into host genome
(+) = can infect ___non diving_________ & dividing cells
(+) = HIV target is T-cells__ so very good for blood disorder
- Disadvantages
- (-) = cannot control where it ___inserts_____, but inserts into essential genes or cancer causing less than retroviruses
How to deliver genes
- 1. Viral vectors
- (A) Retroviruses
- (B) Adenoviruses
- (C) Lentiviruses
- 2. Non-viral vectors
- (A) Liposomes
- (B) Nanoparticles
- (C) Gene pills
- (D) RNAi
How to deliver genes - non-viral vectors
- (A) Liposomes
- Process:
- lipid bilayer__________ vesicle with gene inside
- Fuses with host cell membrane
- Gene endocytosed_________ into cell
- Advantages:
- (+) No size limit_____ of gene to be inserted
- Disadvantages
- (-) does not integrate into host genome
- (-) gene transfer is not as efficient______
- (-) gene expression does not last long
How to deliver genes - nonviral vectors
- (B) Nanoparticles
- Process:
- DNA is a polyanion_____ (due to phosphate group)
- Complexed with polycation______ nanoparticle
- Advantages
- (+) can deliver to dividing & non-dividing cells
- (+) can deliver gene larger than viral delivery methods
How to deliver genes - nonviral vectors
- (C) “gene pills”
- State of the art – only developed in the past 5 years
- A pill delivers the therapeutic DNA
- Pill travels to intestine, DNA is absorbed by cells
- Intestinal cells then express the protein, secrete into blood (D) RNA silencing (RNAi)
- Inhibits mRNA of defective genes
- Can be delivered by plasmid, lentivirus, or inject directly into cell
- RNAi can then bind its complementary mRNA target to inactivate it
- Difficult because RNA is short lived & how to get to healthy vs non-healthy cells
Genetic medicine - gene therapy
- Requirements for approval of clinical trials for gene therapy
- 1. Knowledge of defect/how it causes symptoms
- 2. An animal____ model
- 3. Success in human cells grown in vitro___
- 4. No successful alternative_____ therapies
- 5. safe_ experiments for humans
Gene therapy & Clinical trials
- cancer_____therapies #1 type of clinical trials
- retrovirus_____ #1 type of vector used
- united states______#1 country with clinical trials
- Most clinical trials are in phase 1 ____
Hurdles to gene therapy
- cost___ = treatment is not always permanent & costs $450,000- $1,000,000
- ____single gene only______________= there is not gene therapy for disorders caused by multiple genes
- ethics____ – some gene therapies could breach the Weismann barrier protecting the testes &
affect germline cells
- ____viral vectors________ – can cause toxicity, be attacked by immune cells & inflammatory responses that are dangerous to the patient
- _____not long lasting___________- in some cases the insertion is not stable in the genome & doesn’t integrate properly
- Causes other more _______serious problems__________– if inserted next or within a tumor suppressor gene it could cause cancer
CRISPR-cas 9
- Can now edit parts of the genome by removing, adding, or altering sections of DNA
- Naturally occurs in bacteria to repair viral damage
- Made of 2 molecules
- 1) cas 9_____ enzyme = “molecular scissors” cut DNA in a very specific place
- 2) ___gRNA____ = “guide RNA” is predesigned 20 bases long RNA that guides the Cas9 to the correct place. Is complementary to the DNA that will be cut out
- Advantage = cheap, fast, good results
- Disadvantage = off-target binding/cutting (what if 19 of 20 bases match?)
Stem cells
- All cells arise from the ultimate stem cell = ___fertilized egg___________
- Zygote – first few cell divisions are symmetric = all daughter cells have the same ___potency______ (potential for developing into different cell types)
- After this point cells become :
- 1) committed_______ to pathways to become different cell types &
- 2) ____restricted _______ in their capacity to generate different types of cells
- when a stem cells divides one cell remains a stem cell and the other one differentiates
Stem cell therapy
- allogenic_____ cell therapy = transplanted cells come from ___a donor______
- are genetically different from those cells of the recipient
- high risk of immune rejection, must HLA match
- autologous________ cell therapy = transplanted cells come from themselves________
- avoids immune rejection
- typically involves reprogramming nuclei & gene expression
iPS
- Induced pluripotent stem cells (iPS) = adult cells (ex. skin cells) that are reprogrammed to become late stage_________ embryonic stem cells.
- + can make pluripotent cells that are specific to a _____disease______
- + allows the study the pathway____ of particular disease
- + allows testing ___treatments________ to cure that disease
- + are from the patient so minimal risk of rejection____
- Mesenchymal stem cells = found throughout body
- Can direct to become bone, cartilage, fat, muscle
What can be treated successfully right now
- Currently approved stem cell treatments
- 1) Bone marrow transplants (50 years) – hematopoietic stem cells
- Used to treat blood cancers (leukemia, lymphoma, sickle cell, etc)
- Needle placed into soft center of donor bone marrow or collecting peripheral
blood stem cells by ____ampheresis________ →Collected stem cells transfused into recipient
2) _____umbilical cord____________blood (1988) – hematopoietic stem cells
- Blood that remains in the placenta/umbilical cord after childbirth
- Cannula used to remove blood, stem cells isolated, cryopreserved until needed Stem cell treatments that are the furthest along in clinical trials
• Bone
• Skin
• Cornea
What to warn your patient about
- ____clinical treatment_____________= medical practice that has been shown through formal
clinical trials to be reasonably safe & effective for treating a particular
disease/condition & approved through FDA
- clinical trial_______ = research study to answer specific questions about a new
treatment. Seeks to establish if new treatments are safe & effective. The
treatment is thus far unproven.
- ____expermential intervention__________________ = new, untested, or different from usual medical treatment. Has not been proven that is safe or if it will work in treating disease
Genetic Testing & Its Importance Genetic testing encompasses various methods to determine genetic predispositions, diagnose conditions, and guide treatment. It is categorized into several types:
Preconception Testing: Identifies genetic traits in adults that may affect offspring.
Prenatal Testing: Detects genetic disorders in an embryo or developing fetus, utilizing non-invasive methods like cell-free fetal DNA analysis and invasive procedures such as amniocentesis.
Postnatal Testing: Confirms suspected genetic disorders in newborns or individuals showing symptoms, including newborn screenings for actionable conditions like sickle cell disease and PKU.
Predictive Testing: Assesses risk for hereditary diseases, helping individuals make informed medical decisions.
Carrier Screening: For those with family histories of genetic disorders, helping couples understand risks of genetic conditions in future children.
Forensic Testing: Used for identification purposes in legal settings.
Direct-to-Consumer Testing: Provides genetic information without healthcare provider involvement, but with risks related to privacy and interpretation.
Understanding genetic testing lays the foundation for personalized medicine, enabling targeted treatments and improved health outcomes.