M2M+Unit+II+LOs

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=Pedigree and Mendelian Inheritance=
 * Understand and use standard pedigree symbols.
 * males as squares, females as circles, deceased with lines through them, black as phenotype positive, white as phenotype negative. Diamonds = unspecified-gender kids/fetuses.
 * I, II, III: generations from oldest to youngest). Three generations = complete. Symbols go on the lines.
 * See slide 10 for symbols.
 * Be able to draw a pedigree (elicit family medical, use symbols correctly to draw a pedigree).
 * Be able to interpret a family history.
 * Be able to discuss how the distribution of phenotypes in a pedigree is a reflection of segregation of gene variants (genotypes)
 * I think what he's saying is to be able to say "Hey, this guy with the phenotype had parents who didn't have it. Must be some kind of autosomal recessive disease," or "hey, every time this disease phenotype comes up, at least half the children show it. Must be some kind of autosomal dominant disease," etc. Just a guess though.
 * Recognize the patterns of Mendelian inheritance.
 * Genes come in pairs (sort of- note X-linked diseases in males [only have one X chromosome] and genetic mitochondrial diseases, since mitochondrial DNA is derived exclusively from maternal DNA).
 * Genes come in different versions ('alleles'), which lead to observed phenotypes.
 * Law of Segregation: Alleles segregate at meiosis into the gametes.
 * Law of Independent Assortment: The segregation of each pair of alleles is independent. (exception: genes close together on chromosome, aka linked genes)
 * "Hemizygous": Means you only have one particular gene, not two. Ie: males have a single copy of each X-chromosome gene. Also, presumably, anyone who has only one working copy of a given gene through deletion (or, possibly, imprinting).
 * "Horizontal" patterns of affected phenotype: tends to be **autosomal recessive** (more likely affected in siblings and not in parents).
 * The rarer the disease/alleles, the greater proportion of affected persons will be due to consanguinity.
 * **Autosomal dominant** : tends to appear in every generation; phenotypically normal parents tend not to pass it on to their children. Note new mutations (spontaneous generation of disease) can occur with some frequency.
 * **X-linked recessive** : incidence much higher in __males__ (X-linked __dominant__ disease incidence is higher in __females__). Affected males pass on mutations to **all** daughters and **no** sons. Carrier females' offspring (male and female) have 50% chance of inheriting.
 * Explain how genes ‘segregate’ and apply this knowledge to determining probabilities of inheritance in simple pedigrees.
 * Punnett square, simple Mendelian inheritance: AA x aa -> AA + 2Aa + aa.
 * Understand the factors that affect development of the phenotype in single-gene disorders (roles of: environment, modifier genes, stochastic effects).
 * __Modifier genes__: genetic factors outside a trait's genetic locus that influence that trait's phenotype
 * __Stochastic (random) events__: The "we have no idea" category; hand of God reaching down to give someone cystic fibrosis, or the less blasphemous metaphor of choice.
 * __Phenocopies__: Diseases due to non-genetic (environmental) factors.
 * Explain the terms: penetrance, expressivity, and pleiotropy.
 * **Penetrance** : The fraction of individuals with a disease genotype who show manifestations of the disease.
 * High penetrance: most individuals with genotype get disease
 * Low penetrance: most individuals with genotype don't get disease
 * Can be dependent on age, etc.
 * "Light switch"-- have it (penetrant) or don't (non-penetrant).
 * **Expressivity** : The degree to which a trait is expressed in an individual (ie. severity).
 * "Dimmer"-- how much does a person have the disease?
 * **Pleiotropy** : When a mutation leads to multiple different phenotypes. Notice that this isn't the same as different degrees of the same phenotype (that's expressivity).
 * Define population genetics and use the Hardy-Weinberg principle to estimate carrier frequencies for autosomal recessive disorders.
 * Population genetics: the study of allele frequencies and changes in allele frequencies in populations-- where 'populations' is an arbitrarily defined, but large, number of people.
 * Note that nuclear DNA > 99% similar between human individuals.
 * Notice "polymorphism" refers to any common genetic variant of an allele- that is, it occurs in greater than or equal to 1% of the population.
 * **Hardy-Weinberg principle** : based on the fact that //p// + //q// = 1, where //p// is the frequency of a common allele at a particular locus and //q// is the frequency of a rare allele at that locus.
 * If you square the equation, you can get a nice equation for dealing with frequency of homo- and heterozygous phenotypes in a population: **//p//** **2** **+ 2** **//pq//** **+** **//q//** **2 = 1.**
 * Notice that you calculate //p// or //q// by dividing the number of **genes** (each individual has 2 genes, each of which may be affected or not) by the total number of **genes** in the population.
 * Notice that you calculate //p// 2, 2 //pq//, or //q// 2 by dividing the number of **individuals** who fit that genotype [usually assessed by their phenotype] by the total number of **individuals** in the population.
 * This makes it possible to calculate the autosomal recessive carrier population (2//pq// ) given the number of affected ( //q// 2) individuals.
 * Example: You've got two individuals in a population of 10 that are affected by an autosomal recessive disease. 2 individuals with homozygous recessive (//q// 2) out of a population size of 10: //q// 2 = 2/10 = 0.2. Taking the square root of that, //q= 0.45. Thus p= // (1 - 0.45) = 0.55. So //p// 2 = 0.3 and 2 //pq// =0.5. Thus you can say of our small population that the number of homozygous dominant individuals (no autosomal recessive genes) is (0.3 * 10=) 3, and the number of heterozygous (carrier) individuals is (0.5 * 100=) 5.
 * Notice that if a disease is very rare (1/10,000 or rarer), 2//pq// can approach 2 //q//, since //p// is pretty much equal to 1 (100% frequency).
 * Use basic principles of population genetics (mutation rates, fitness, effects of consanguinity, addition of new mutations to gene pool).
 * Notice that Hardy-Weinberg doesn't usually work perfectly-- non-random mating, mutation and selection pressures, small population size, and medicinal palliative effects on affected individuals throws off the H-W equilibrium.
 * If a disease is recessive, it's less affected by fitness and selective criteria.
 * Notice this in the lecture notes: "By observing the number of cases of disease in a population it is possible to calculate __rates of mutation__ for different conditions."
 * What this means: for an autosomal dominant disease, the number of new conditions with no previous family history of the condition can be regarded as the number of new gene mutations causing that condition.
 * Notice that the math here is about **genes**, not **individuals** :
 * So if there's 5 new cases of X disease in 100 live births, that means 5 newborn genes out of 200 (100 newborn individuals with 2 genes each) have the mutation.
 * The symbol for the mutation rate for a given disease is **mu** ( μ ).
 * 5 genes with the mutation divided by 200 total newborn genes = μ =(5/200)=0.025.
 * Also can express μ as simply 5 mutations in 200 or 1 in 40.

=Genome organization=
 * Know what proteins human genomic DNA combines with, what they are likely to do, and how they are arranged with human DNA
 * They combine with histones/nonhistone proteins. DNA + histones/nonhistones: chromatin.
 * Four core histone subunits: H2A, H2B, H3, H4, each with two copies (make up a histone octomer all together).
 * Roughly 140 bp wrapped around a given histone; roughly 60 bp connecting one histone to the next. Thus __200 bp = repeating unit of DNA__ per histone.
 * Know the terms associated with various DNA/protein structures mentioned in the above outline, e.g. nucleosome, chromatin, etc.
 * (nucleosome = histone + DNA complex)
 * Know the various structural levels of organization of genomic DNA/protein complexes as described in the above outline:
 * Double helix: DNA
 * "Beads on a string": DNA wrapped around histones (nucleosomes)
 * "Solenoid": Nucleosomes wound into long cylinder
 * Aggegrations of solenoids as 100 kbp [1 __k__ilo-__b__ase __p__air = 1,000 base pairs. Often notated as "kb" instead of "kbp."] loops attached to nonhistone scaffolds
 * Know that in addition to a nuclear genome, human cells contain the mitochondrial genome
 * Mitochondrial DNA inherits solely through the mother
 * Know the features related to the mitochondrial genome described in the following outline
 * Mitochondria: ~ 16 kbp: a few dozen genes
 * Uses slightly different codon code than nuclear DNA
 * Notice that most proteins acting in mitochondria are made by nuclear DNA instead.
 * Know the fundamental principles regarding the evolution and organization of the human genome mentioned in the outline
 * Genome is both a trace of past evolution (shows various evolutionary pressures) and a base for future evolution.
 * Roughly 30 new mutations occur in each individual.
 * Thus- there isn't "a" human genome, any more than there's "a" human fingerprint.
 * Know that genome variation is an essential fuel of evolution and adaptation, but it also produces human disease.
 * See next point.
 * Know that random variation, when it has an effect, is almost always harmful
 * Given the absurd degree of complexity in protein-protein interaction, this makes sense. Also gives you a sense of the amount of time that evolution [ie beneficial mutation trends] tends to require.
 * Know the dynamic nature and non-random organization of the human genome as described in the outline
 * Gene-rich and gene-poor, stable vs unstable regions, GC-rich vs AT-rich, euchromatic and heterochromatic regions (see below).
 * Notice that, in general, the location of genes in a given gene family in the chromosomes are not related to each other.
 * Notice that chromosomes are numbered by size (#bp) but the number of __genes__ encoded by each chromosome doesn't correlate with its size.
 * Know how frequently a SNP is likely to occur between two individuals
 * **SNP** : Single nucleotide polymorphism (difference is a single base pair)
 * Average: 1 SNP per 1000 bp
 * This means 99.9% identical between individuals
 * Also means roughly 3,000,000 differences between individuals (1/1000 of three billion bp in the human genome)
 * Know the types of variations that occur between genomes
 * Insertion-deletion polymorphisms:
 * Minisatellites: tandemly repeated 10-100 bp blocks of DNA (highly variable number).
 * Here, labeled as **VNTR** (variable number of tandem repeats), though notice the Wiki for this has both mini- and microsatellites as subsets of VNTRs.
 * Notice VNTR can be used as individual genetic fingerprinting.
 * Microsatellites: 2-,3-,4-bp repeats (> 50,000 per genome). Also known as short tandem repeat polymorphisms (**STRP** s).
 * The point, I think, is that you'd expect a lot of the variation between individuals to come from variations in these large blocks of small repeating segments, instead of variations between important genes.
 * Single Nucleotide Polymorphisms (SNPs):
 * Frequency of ~ 1 per 103 bp.
 * Can be detected by PCR and used for genetic fingerprinting.
 * Copy number variation
 * Variance in the number of copies in a particular gene in an individual.
 * Variation in size of segments of genome copied: ranges from 200 bp to 2 Mbp [1 __m__ega-__b__ase __p__air = 1,000,000 base pairs. Also abbreviated Mb.].
 * Know the points listed in the outline related to the following: a) gene-rich, b) gene-poor, c) stable, d) unstable, e) GC-rich, f) AT-rich, g) euchromatic, h) heterochromatic
 * There are "gene-rich" regions or chromosomes (ie Chr 19) and "gene-poor" (ie Chr 13, 18-21), corresponding to how many genes (expressed segments) each contains.
 * Most of genome is classified as stable (ie doesn't change/mutate a lot). Of the remainder that's unstable, a lot of its variation is related to diseases.
 * GC-rich (38% of genome) vs AT-rich (54% of genome): pattern of GC/AT-rich areas is not random.
 * **Euchromatic** regions: more relaxed, less repeats. Makes up most of the genome.
 * **Heterochromatic** regions: more condensed, more repeats. Tends to be near centromeres and makes up less of the genome.
 * Know that genome sequencing efforts are focused on euchromatic regions
 * Right.
 * Know that many sequencing gaps still remain even in euchromatic regions
 * It's not a completely finished project yet.
 * Know to what categories genomic DNA sequences can be assigned as described in the outline; know the frequency of each class
 * **1.5%** = translated (ie exons that code for protein)
 * **20-25%** = genes (introns, exons, UTRs, gene regulating factors)
 * Notice the vast majority of "genes" are not translated.
 * **50%** = "single copy" sequences (one copy of gene)
 * **40-50%** = "repetitive DNA" (a sequence that's repeated 100-1,000,000 times)
 * As described in the outline, know the types, locations and frequency of repetitive DNA sequences that exist in the human genome
 * Tandem repeats: "Satellite DNA"
 * Seems to be part of the basis for the differential dye binding that's used in cytogenetic chromosome banding (ie G- or M-binding, see "Cytogenetics").
 * Some found in heterochromatic regions
 * __Alpha-satellite repeat__: 171 bp repeat unit found near centromeric region of all human chromosomes.
 * Dispersed repeats
 * These are retrotransposon elements in the DNA. Effectively they can copy their own sequences into other locations in the DNA.
 * Having worked in a lab looking into these, there's some thought that they're some kind of foreign DNA using our DNA to propagate itself, like mitochondrial DNA but less clearly symbiotic.
 * Short Interspersed Nuclear Elements (SINEs) (aka Alu family):
 * 500,000+ copies in genome (Wiki says up to 13% of human genome, which is considerably more than that), ~300 bp per copy.
 * Long Interspersed Nuclear Elements (LINEs) (aka L1 family):
 * 100,000+ copies in genome (Wiki says more like 900,000), ~6 kbp per copy.
 * Can be medically relevant: retrotransposition of copy in the middle of another gene may inactivate that gene.
 * Know the estimated number of human genes, and the different types of genes as described in the outline
 * The gene number = 25,000-30,000; comprised of:
 * protein-encoding genes
 * RNA-encoding genes
 * "pseudogenes": non-functional but homologous copies of existing genes.
 * Split up into "intron-containing" (presumably a gene duplication that's inactive for lack of a promoter region) or "intronless" (presumably a segment of transcribed, processed DNA that's been retrotransposed into the genome).
 * Know that genes can exist in families and gene families arise by gene duplication
 * Gene family: genes with high (>85%) sequence similarity. Perform similar functions.
 * Know the advantage of gene duplication as an evolutionary mechanism
 * When a gene duplicates, it frees one copy to vary while the other copy continues to perform its function.

=Clinical vignettes=

Down's syndrome
The most common chromosomal abnormalities in newborns (others are often lethal) Polyhydramnios: excess of amniotic fluid (baby not able to swallow fluid) Points on handout:
 * Advanced maternal age (no prenatal care – can discuss what tests might have led to suspicion of DS prenatally had mother received prenatal care)
 * Down's syndrome and trisomy incidence rises with the age of the mother ("advanced" = 35 or older): risk of DS baby at 35 is 1:200. (at 40, 1:100- exponential increase in risk after 35)
 * Karyotypic analyses:
 * Amniocentesis to look at baby's chromosomes is routinely offered to expecting mothers of advanced maternal age. Done in early 2nd trimester. Needle into amniotic fluid, picking up shed skin cells. Process takes about 1.5-2 weeks.
 * Chorionic villous sampling: Take fetal tissue from where fetus attaches to uterine wall. Results much quicker; done after 11 weeks (1st trimester).
 * Fetal ultrasound: look for fat neck, short femurs
 * Blood screens in mother: looking for fetal blood markers
 * Risk of a person having a second trisomic baby after having a previous one: about 1:100.
 * Typical phenotype of DS, including hypotonia:
 * Flattened back of head (occiput) : brachecephaly
 * Midface hypoplasia (incomplete midface development)
 * Epicanthal folds (folds at corners of eyes)
 * Ears (pinnae) small and set low in head
 * Bilateral transverse palmar creases
 * Accentuated space between 1st and 2nd toes
 * Decreased muscle tone (hypotonia)
 * Abnormal tooth development
 * GI tract problems (mainly esophageal/duodenal atresia: ~ 15%)
 * Notice that the tongue is normal-sized in a too-small cavity.
 * Congenital heart disease (can discuss incidence, and what are the most common lesions)
 * Diagnosed with ultrasound/echocardiogram
 * Between 1/3 and 1/2 Down's syndrome babies are born with congenital heart disease (septal defects, etc).
 * Common heart problem: atrioventricular canal (hole between all four heart chambers, requires surgical repair).
 * Notice that heart conditions of babies with other trisomies are not routinely repaired.
 * Esophageal atresia – discuss GI tract atresias in DS, polyhydramnios, need for immediate surgery
 * Esophageal atresia: esophagus ends in blind pouch (no ability to swallow amniotic fluid, leading to polyhydramnios). Obviously needs to be able to swallow, thus need for surgery.
 * Genetic testing – timing of results for karyotype and FISH (more impt for other trisomies)
 * FISH usually used looking for trisomy; results within hours. (note trisomy isn't strongly correlated with Down's syndrome)
 * Karyotyping: see above (amniocentesis, chorionic villous sampling)
 * Trisomy characteristics:
 * Classically, trisomy on chromosome 21 is associated with Down's syndrome. Sometimes, chromosome 21 gets stuck on other chromosomes as well-
 * Some DS patients have 21 trisomy in //all// of their cells, but some only have it in //some// of their cells. This latter condition is called **mosaic trisomy** ; people with it tend to be on the higher-functioning end of the curve that those with trisomy in all their cells.

Prader-Willi syndrome

 * Presentation in newborn period with hypotonia and dysmorphic features, undescended testicles.
 * Diagnosis made with FISH – will further discuss importance of particular region of 15q during presentation
 * Notice that Prader-Willi and Angelman Syndromes both occur due to irregularities with the long arm of chromosome 15.
 * Early course of failure to thrive and feeding difficulties which reverses during preschool age when children develop hyperphagia and gain weight
 * Driven by hypothalamic change?
 * Treated with growth hormone: prevents development of obesity, promotes height
 * Mild-moderate developmental delay leading to mental retardation as adults
 * Ophthalmologic problems common – especially strabismus and nystagmus
 * strabismus: "lazy eye" condition
 * nystagmus: "jiggly eye" condition

Thalassemia

 * Notice HbS, HbC, and HbE: mutant forms of hemoglobin (caused by beta globin mutations).
 * S form: common in __east Mediterranean__ and __Africa__. [sickle-cell]
 * C form: common in __Africa__
 * E form: common in __SE Asia__ and __west Pacific__
 * Thalassemia common (most to least): SE Asia, Africa, west Pacific, east Mediterranean
 * see handout for case history
 * Swollen stomach can be due to hemoglobin production in liver and spleen after birth
 * "SS" disease: sickle cell disease.
 * "Hemoglobin H": HbH disease, compound heterozygosity; one allele has no alpha globin production, the other has impaired alpha globin production. Usually found SE Asia. Doesn't usually result in hepatosplenomegaly.
 * MCV = "Mean corpuscular volume": size of red blood cells.
 * "Reticulocytes": young red blood cells (sign of increased RBC production).
 * Erythroblasts: red blood cell precursor. Usually found in marrow- not normally found in blood.
 * Notice an excess of alpha cells can bind to and rupture red blood cell membranes. This is why, clinically, you see bilirubin buildup and result in a jaundiced appearance.
 * Nice summary of beta-thalassemia syndromes on the handouts, p. 8.

Von Hippel-Lindau

 * Identify the typical manifestations of von Hippel-Lindau (VHL) disease and recognize their vascular nature.
 * A familial cancer syndrome, manifesting mainly in vascular tumors.
 * Constellation of CNS/retinal hemangioblastomas, renal cancer, pheochromocytoma (tumors on adrenal cells)-- although some families have the adrenal tumors and some don't.
 * Uncommon: 1/36,000 births. Inherited in an autosomal-dominant fashion, though it's a heterozygous germ-line mutation.
 * Common causes of death: CNS hemangioblastoma (formerly), renal carcinoma (currently).
 * Understand the role pVHL plays in the regulation of Hif 1-alpha and the consequences of mutations in VHL.
 * Hif 1: hypoxia-induced factor 1. Controlled transcription of genes related to hypoxia.
 * [Gene:] VHL disease shows abnormality on short arm of chromosome 3: common deleted region, which codes for pVHL (VHL protein).
 * Proteins that surround pVHL target other proteins from degradation (ubiquitinylation).
 * Normally, pVHL, in the presence of oxygen, targets Hif 1 α for degradation. At low O2 levels, the Hif 1 α protein changes conformation and is no longer recognized by pVHL complex (thus turns on anti-hypoxia genes).
 * In VHL disease, when you lose pVHL, the Hif 1 α protein isn't degraded by pVHL and is thus constantly transcribing anti-hypoxia genes even when O2 supply is adequate.
 * One of the things this results in is an uncontrolled transcription of vascular endothelial growth factor (VEGF), which bind to its receptor (VEGF-R) to cause carcinogenesis in the vascular cells.
 * Timing of tumor development in familial VHL disease seems to support LOH (see "Molecular basis of carcinogenesis") and Knudson's 2-hit hypothesis.
 * Recognize that VHL is sporadically mutated in many cases of renal cell carcinoma that are not associated with the inherited von Hippel-Lindau disease.
 * 60-70% of sporadic (not familial VHL) clear-cell kidney cancers have inactivation of both VHL alleles-- common cause (which means it's an important gene in vascular kidney tumors).
 * Recognize that recently approved biologically targeted agents to treat renal cell carcinoma are based on the pathophysiology of VHL mutation.
 * Little success in treating Hif 1 α levels.
 * Successful treatments are based on VEGF - vascular endothelial growth factor.
 * Monoclonal antibody against VEGF (not particularly effective in kidney cancer).
 * Also tyrosine kinase inhibitors against VEGF-R: these seem to be more effective.
 * Notice that VEGF-R is crucial to developing //new// blood vessels-- thus this treatment is not necessarily going to do anything about existing vascular tumors. May become a game of managing existing tumors rather than eradicating them.

Diabetic Ketoacidosis (DKA)

 * Summary:
 * Basically, during uncontrolled diabetes, the cells can't get enough glucose inside them to make sufficient energy to survive-- effectively, the cells are starving because there's no insulin (or no effective insulin) getting the glucose into cells and out of the bloodstream. Thus **diabetic** ketoacidosis.
 * Because the cells need energy and they can't get it from glucose, they begin to break down other substances, like muscle proteins and stored fat, for energy. One of the byproducts of non-glycolytic energy production (lipolysis) is ketone bodies (thus **keto** acidosis), which accumulate more rapidly than they're able to be disposed of.
 * Notice that the 'fruity' odor on the breath of DKA patients results from the high levels of ketone bodies in the blood.
 * The accumulation of circulating ketone bodies lowers the pH of the blood (thus keto**acidosis** ). Lowered blood pH can break down the proteins of tissues throughout the body, leading to multiple organ failure.
 * More to the clinical diagnostic point, when the blood pH goes down, the breath patterns get very different (deeper) in an effort to clear CO2 from the blood (remember CO2 makes blood more acidic).
 * Dehydration frequently results, even with good fluid intake, because the kidneys aren't able to keep up with all the excess glucose in the blood, and some glucose leaks out into the urine. This draws a lot of water with it and the patient becomes dehydrated.
 * Generally treat DKA with insulin-- corrects acidosis, which allows the true K+ levels to be seen in the ECF-- so can treat the low K+ levels accurately at that point. Also treat with fluids to correct dehydration.
 * Review insulin release:
 * DKA usually hits type I diabetics, less frequently type II diabetics.
 * Insulin is released during food consumption.
 * Promotes uptake of glucose into cells, inhibits formation of glucose in the liver, begins production of glycogen as a storage mechanism.
 * Review pathophysiology of DKA:
 * Notice that diabetes often occurs along with other autoimmune diseases (like hypothyroidism) in family histories.
 * Rapid breathing, nausea, vomiting, thirsty abnormally high urine output.
 * Elevated glucose, low venous pH, high potassium levels in blood.
 * Understand the evolution of potassium during the development of initial treatment of DKA.
 * In DKA, as the [H+] in the ECF goes up, the H+/K+ exchangers take H+ into cells and pump K+ out of them; this results in total body K+ depletion even though extracellular levels of K+ are high.
 * Describe the risk of cerebral edema during treatment of DKA:
 * Cerebral edema: shows as sluggish, combative behavior and headaches. Major source of mortality in DKA, also can result in significant neurological damage.
 * Cerebral edema results in part from the fact that, after the administration of insulin, the cells in the brain uptake glucose much faster than the rest of the body. This means that the brain cells have a high concentration of glucose relative to the blood-- and osmotically, water travels down that gradient out of the blood into the cells, which expand and cause edema in the brain.
 * This is treated by mannitol-- this raises the concentration of the ECF in all of the body aside from the brain (doesn't cross the blood-brain barrier), which helps equalize the solute concentrations so that the osmotic gradient doesn't flow into the brain cells.

Multiple Sclerosis

 * MS: disease of CNS: spares peripheral NS.
 * progresses either as attacks of symptoms that come and go (85%) or a time-progressive course (15%).
 * See lots of different progression charts in powerpoint. Very variable course.
 * Notice that acute attacks locally break down the blood-brain barrier.
 * Autoimmune disorder: immune system attacks CNS and demyelinates axons.
 * Hallmark: slowing of axon conductivity. However, does also cause axon loss.
 * 75% of new cases present between 15-45; 5% are under 18; mostly women (2/3) and whites. Most common inflammatory CNS disease. Gets more common away from equator.
 * Characteristic lesions on axons: round or ovoid, next to ventricles, optic nerves, spinal cord, etc. These remain but become less inflamed over time.
 * Possible causes: related to DR2 and IL-7 genes; increased risk in mono- vs. dizygotic twins; Apo E e4 homozygosity; chlamydia and various viruses (epstein-barr), smoking, sunlight/vit D.
 * Diagnosis: CNS lesions disseminated in space (two distinct loci), time, objective abnormalities on neuro exam, no other cause can be identified.
 * Symptoms: numbness, tingling, loss of or abnormal vision, weakness, balance, urinary urgency, constipation; later on, fatigue (main symptom), sex dysfxn, gait dysfxn, depression, pain, dysphagia.
 * Tests: MRI, CSF, biopsies, evoked potentials (testing conduction velocity with visual/brainstem/auditory/somatosensory stimulation)
 * CSF: abnormal immunoglobulins (**oligoclonal bands** on gel), but rarely high WBC.
 * Treatment of acute attacks: IV methylprednisolone, 1g/day x 3-5d, or plasma exchange once a day for about 2 weeks if steroids don't work.
 * Can also suppress or modulate immune system:
 * Modulate with interferons (reduce attack rate by 1/3, slow progression).
 * Can also use natalizumab (reduce attacks and new lesions substantially but is assoc. with fatal brain infections).
 * Demyelination of axons produces proliferation of Na+ channels down axon- slows nerve conduction, can lead to calcium influx. Can use Na+ channel blockers to treat.
 * Note that demyelination symptoms get worse under high heat.

=Chromosomal Abnormalities=
 * Describe the events in meiosis that produce genetic variability among offspring.
 * ["Mosaicism": abberant process in which cells with two different genetic complements are produced during mitosis after the zygote is already formed. (Doesn't occur during meiosis, but often cells will reproduce mitotically before meiosis, thus this affects meiosis as well.)]
 * Meiosis:
 * Chromosomes replicate into sister chromatids (identical chromosomes).
 * Meiotic recombination: homologous crossing-over occurs between non-sister chromatids.
 * Recall that only one of the four haploid products of female meiosis actually becomes an egg; the rest atrophy into polar bodies.
 * About meiotic recombination:
 * Each cross-over is called a **chiasmata**.
 * These cross-overs have a structural purpose: they steady the pairs of homologous chromosomes next to each other so that division can occur smoothly between them. This implies that successful meiosis can't occur without crossing-over recombination (which in turn makes recombination more frequent).
 * Cross-overs also occur between X and Y chromosomes in male meiosis.
 * This results in a reshuffling of genes between chromosomes in meiosis
 * Compare and contrast mitotic and meiotic cell divisions.
 * Mitosis in somatic cells and primordial germ cells; meiosis exclusively in germ cells
 * Mitosis: two diploid products; meiosis: four haploid products
 * Mitosis: two identical genetic products; meiosis: shuffled or randomized genetic products
 * Mitosis: centromeres divide at anaphase; meiosis: centromeres divide not at the first but the second anaphase (in splitting the sister chromatids)
 * Mitosis: one S (DNA replication) phase per division; meiosis: one S phase for both divisions.
 * Describe the relationship between meiotic recombination (cross-overs) and chromosome nondisjunction and differentiate between the reproductive consequences of nondisjunction events in meiosis I versus meiosis II.
 * Recombination is a normal swapping of genetic material between homologous chromosomes in meiosis I before separation. Nondisjunction events are abberant and result in an uneven distribution of genetic material when the cell divides.
 * Nondisjunction event in meiosis I: both pairs of sister chromatids go to one daughter cell after first division. This results in one quadroid (4 chromosomes) daughter cell and one daughter cell with no genetic material at all. When meiosis II occurs, you wind up with two diploid cells and two nongenetic cells, none of which are correctly functional as products of meiosis.
 * __Nondisjunction in meiosis I__: All daughter cells dysfunctional.
 * Nondisjunction in meiosis II: meiosis I progresses normally, with the homologues separating as they should. However, in meiosis II, one pair of sister chromatids fails to separate, resulting in two normal haploid meiotic cells, one abnormal diploid cell, and one cell with no genetic material.
 * __Nondisjunction in meiosis II__: Half of daughter cells dysfunctional.
 * Understand and interpret the karyotypic designations used in clinical genetics reports to describe both numerical and structural chromosomal abnormalities.
 * (A lot of stuff he talked about can fit under this rubric:)
 * Giemsa: dye used to create banding patterns in chromosome based on selective binding. (ie. "G-banding," see "Cytogenetics .")
 * Ideogram: depiction of the banding pattern of chromosome, with the bands numbered //outwards// from the centromere. Notice bands 11 and 12 are called "one-one" and "one-two" respectively (as opposed to normal-people talk "eleven" and "twelve").
 * There are three types of chromosome structures based on the position of the centromere relative to the two arms (centromere also called "primary constriction"):
 * **Metacentric** : The centromere is in the center of the chromosome.
 * **Submetacentric** : The centromere is offset from center, producing a longer and a smaller arm.
 * **Acrocentric** : The centromere is near one end of chromosome; there's a small nubbin of chromosome above it (contains " **stalk** " with ribosomal rRNA-producing DNA, and " **satellite** " region above this) and a very long arm below.
 * "**p** " = short arm of chromosome (petite)
 * "**q** " = long arm of the chromosome
 * Notice that in metacentric chromosomes, this designation is conventional, but arbitrary.
 * "Proximal" or "distal" designations are relative to the centromere.
 * **Ways to notate chromosomal abnormality** [he spent a lot of time hyping this, so pay close attention to it]:
 * General format: (number of total chromosomes),
 * types of mutation notations:
 * Addition of chromosome (+)
 * Deletion (del)
 * Inversion (inv)
 * Duplication (dup)
 * Insertion (ins)
 * Ring (r)
 * More in Thompson + Thompson p. 66
 * examples:
 * 46, XX del(5p) = normal number chromosomes, female, deletion in chromosome 5, petite (short) arm.
 * 47, XX +21 = one additional chromosome, female, additional copy of chromosome 21 (ie Down's Syndrome).
 * 46, XY, del(4)(p16.3) = normal number chromosomes, male, deletion in chromosome 4, on band 16.3 on the petite arm.
 * **Aneuploidy** : loss or gain of selected chromosomes (usually fatal but note below about clinical trisomies).
 * Often due to selective problems with meiotic disjunction.
 * Specifically, due mostly to maternal meiosis I disjunction.
 * **Polyploidy** : extra copies of all chromosomes (triploidy, tetraploidy); almost always fatal.
 * Often due to complete (all chromosome) meiotic disjunction: can be result of two sperm fertilizing an egg; can also result from sperm fertilizing a diploid egg or a diploid sperm fertilizing an egg (due to nondisjunction event during egg or sperm meiosis).
 * **Mosaicism** : When the zygote contains two cell lines differing in chromosome number.
 * Post-zygotic **mitotic** event results in chromosomal abnormality
 * Affects various tissues depending on nature of abnormality
 * Can be polyploid mosaic or aneuploid mosaic
 * Generally less severe than complete aneu/polyploidy
 * Describe the clinical features of common human trisomies: 21, 18, and 13.
 * 21: __Down's syndrome__: most common survivable trisomy
 * Congenital heart disease
 * Hypotonia
 * GI abnormalities
 * Early onset Alzheimer's disease
 * Note mosaicism (incomplete 21 trisomy)
 * 18: __Edwards syndrome__
 * Congenital heart disease
 * Hypertonicity (clenched hands, narrow hips)
 * Severe CNS abnormalities/retardation
 * 13: __Patau syndrome__
 * Most clinically severe of the 3
 * CNS abnormalities
 * Omphalocele (herniation of GI organs outside abdomen)
 * Renal dysplasia
 * Congenital heart disease
 * Other notes:
 * Abnormalities in sex chromosomes tend to be less severe than in autosomal chromosomes.
 * Klinefelter syndrome (47, XXY) : tall, hypogonadism, breast tissue, frequently infertile, language/reading retardation.
 * Often due to a defect in paternal, as opposed to maternal, meiosis I.
 * about 15% due to mosaicism (dysfunction in a post-zygotic mitotic division)-- zygote okay, problem occurs afterwards.
 * 47, XYY:
 * Often due to a defect in paternal meiosis II
 * delayed speech and language skills, behavioral problems
 * Turner syndrome (45, X) :
 * Webbed neck, swelling of hands and feet, renal, cardiovascular abnormalities
 * Mainly spontaneously aborted
 * about 25% due to mosaicism

=Biochemical Basis of Genetic Disease=
 * Understand at a molecular/gene level those sites where mutation can occur, and how these specific mutations can affect the normal gene product.
 * Missense/nonsense mutations within exons can affect structure of proteins
 * Mutations in promoter/enhancer elements can affect rate of transcription
 * Mutations at cap site or poly-A consensus site can affect stability of transcript and translation
 * Mutations can occur pretty much everywhere, seems to be the take-home message, and can result in pretty much every bad thing you can think of.
 * Specifically, lots of mutations have been identified in __hemoglobin__ and __glucose-6-phosphate dehydrogenase__.
 * Understand why many biochemical variants are not deleterious. Appreciate the number of mutant lethal genes we all carry in our genomes.
 * (1) Degeneracy of the genetic code: change in base pair doesn't necessarily give rise to a different amino acid.
 * (2) If one amino acid is changed for a similar one (basic polar to basic polar, aromatic for aromatic, etc), often there's not a noticeable change in protein function.
 * (3) Amino acid replacement at a non-structural or non-catalytic site may not affect protein functions.
 * (4) Diploid nature of genes means that we automatically have a backup.
 * This also means that most of us are carrying around lethal mutant genes that don't kill us because we rely on the working, 'spare' copy of the gene.
 * Compare dominantly and recessively inherited diseases in terms of their mutant gene products. Understand when a particular mutation will be expressed in a dominantly inherited fashion.
 * Recessively inherited diseases: need both genes to be bad before a disease phenotype crops up.
 * Dominantly inherited diseases: only need one bad copy of a gene to produce disease or death.
 * __Dominant inherited disease mutations tend to be in genes coding for__:
 * Structural proteins
 * Disease: **Osteogenesis imperfecta** (improper collagen assembly, often fatal)
 * Regulatory proteins
 * Disease: Waardenburg syndrome (mutation in trans. factor PAX3)
 * Proteins which induce growth or development
 * Disease: Holoprosencephaly (mutation in Shh [sonic hedgehog] gene)
 * Enzymes whose concentration determines the rate of reaction
 * (normally enzymes are at saturating levels in cells)
 * Disease: **Acute intermittent porphyria** (insufficient levels of urophorphobilogen synthase, which enables formation of heme groups)
 * Recessive inherited disease mutations tend to be in genes coding for __proteins that are normally present at a surplus level__ in cells.
 * Understand the concept of "compound heterozygote", and how it is related to the variable phenotype of many autosomal recessive disorders.
 * **Compound heterozygotes** : inherit two different mutant alleles; display variable severity.
 * Ie: I get one copy of XYZ gene from Mom with a nonsense mutation in its middle (thus that gene produces half-length XYZ proteins). I get another copy of that gene from Dad with a nonsense mutation right at the beginning (thus that gene produces no XYZ proteins). I don't have the exact same mutation in both genes (thus not exactly "homozygous" at that allele) but since I have reduced protein capacity from both genes, I probably have the disease, even if it's recessively inherited. Notice that if my brother had similar mutations in both genes, but near their ends, he would probably have more functional copies of the protein than I would (and thus have a less severe manifestation of the disease), even though we're both lumped in as "compound heterozygous."
 * Understand how genetic defects in regulatory proteins, transport proteins, receptor proteins, and structural proteins can give rise to disease.
 * Transport:
 * **Cystinuria** : Defect in cysteine transport protein results in crystallization of cysteine in bladder
 * Cystic fibrosis
 * Receptor:
 * **Hyperlipoprotein II** : defect in LDL (low-density lipoprotein) receptor which picks up cholesterol and transports it into cells. Results in hypercholesterolemia-- cholesterol accumulates in the plasma (thus leads to cardiovascular disease).
 * **Androgen Insensitivity syndrome** : we've seen this before (mutation in androgen receptor zinc finger DNA binding region).
 * Structural:
 * **Hereditary spherocytosis** : mutations in structural elements of red blood cells. This forces the cell to lose its tight disc shape and assume a fragile spheroid form. This sticks inside the spleen, gets lysed, and leads to anemia.
 * Cofactor interaction with apoenzyme ("regulatory" protein?):
 * **Cystathionuria** : Km defect (see below) in which the cystathionase enzyme is altered and cystathionine is excreted in the urine. Can be treated with an excess of vitamin B6.
 * Homocystinuria: Similar; leads to accumulation of homocysteine in the urine due to a mutation in the appropriate enzyme. Treated the same way.
 * Catalytic defect in biosynthetic pathway:
 * **Orotic aciduria** : Anemia and a buildup of orotic acid due to a mutation in pyrophosphorylase and decarboxylase enzymes. Effectively, can't make new nucleic acids. Treated with uridine.
 * Understand the concepts: “dominant negative mutation”, “gain of function” mutation, “novel function” mutation, and “Km defect” mutation.
 * **Km defect** : mutation where an enzyme's affinity for substrate or for its cofactor are altered (thus raises Km, amount of substrate needed for cellular function of the enzyme).
 * **Dominant-negative mutations** : occur when the mutated gene product adversely affects the non-mutant gene product.
 * **Gain of function** : interchangeable term for " **novel function mutation** ". They both mean effectively that the gene product gets a new, abnormal function.

=Cytogenetics=
 * Recognize the landmarks of human metaphase chromosomes and know which specimens yield different levels of band resolution
 * (good to know that metaphase is when you're generally looking for chromosomes.)
 * short arm, centromere, long arm, telomere at ends. In acrocentric chromosomes, also have stalks (containing ribosomal genes) and satellites
 * Types of chromosomes: metacentric, submetacentric, acrocentric (see "Chromosomal Abnormalities").
 * "G-banding" = current normal process for dye-banding chromosomes. Essentially you use a dye that binds to various regions of DNA and not to others, creating a characteristic "banding pattern" on each chromosome. Good for diagnosing gross problems (ie loss or translocation or chromosomes) but not good at detecting small deletions (since the deletion makes up a tiny fraction of the chromosome and its loss can't be readily observed). You need FISH to detect microdeletions.
 * **G-light bands** : GC-rich (chromatin more compact), contain a lot of short interspersed repeat elements, replicated early, rich in transcribed and housekeeping genes.
 * **G-dark bands** : AT-rich (chromatin more open), contain a lot of long interspersed repeat elements, replicated late, have sparse genes and simpler sequence, tend towards tissue specific genes (heart, liver, etc) as opposed to tissue-universal genes.
 * Number of bands/lengths of chromosomes differ between tissue samples:
 * Very short to short chromosomes [fewer bands] found in:
 * -bone marrows
 * -solid tumors
 * Have 300-400 bands/haploid chromosome
 * Short to medium chromosomes [more bands] found in:
 * -chorionic villous sampling
 * -amniotic fluid
 * -products of conception (don't ask me what this means)
 * -some newborns
 * Have 400-550 bands/haploid chromosome
 * Medium to long chromosomes [most bands] found in:
 * -peripheral blood
 * -mitogen (DNA replicating agents)-stimulated tissue
 * Have 450-650 bands/haploid chromosome
 * Delineate types of FISH probes and how they compliment standard cytogenetic analysis
 * Note she only mentions the first three in the handout.
 * Centromere FISH probes: "**cen** " probes
 * Used for enumeration of chromosomes (prenatal diagnosis of trisomy, etc)
 * Locus-specific probes : "**LSI** " probes
 * Used for detecting deletion/duplication of genes (loci)
 * Dual fusion/fusion: "**DF** / **F** " probes
 * Used for detecting translocation
 * Break apart: "**BAP** " probes
 * Used for detecting rearranged + translocated chromosomes
 * Whole chromosome paint: "**WCP** " probes
 * Used for identifying markers or translocations
 * **FISH** [Fluorescent //in situ// hybridization]: Method to examine subtle deletions or changes in chromosomes that may not be picked up by banding patterns alone:
 * Small deletions
 * Test host vs donor marrow cells after a transplant
 * Can use FISH to look at a large number of cells at once
 * Usually done after preliminary chromosome dye banding
 * Identify which chromosomes can be carried by patients in a mosaic state and what is the implication for the clinical phenotype
 * Recall: mosaicism is two distinct cell lines in an individual (one normal, one abnormal).
 * Chromosomes for which mosaicism is viable: **8, 13, 18, 21** (last 3 most common)
 * Often mosaicism results in a partial or incomplete pathophenotype.
 * Characterize the laboratory test algorithm for children who present with learning disorders, developmental delays, dysmorphic features, and/or failure to thrive
 * First, run a standard high-res chromosome banding analysis.
 * If abnormal, check parents' chromosome banding.
 * If normal, run aCGH (see below) to check for gene deletions/duplications.
 * If one's detected, look into database of genetic variants to match it up with a disease syndrome. Can check parental chromosomes with FISH if there's ambiguity.
 * Clinical note: trisomy 21 can be 'free trisomy' (extra free-floating chr. 21), translocation (extra chr. 21 stuck on another chromosome), or mosaic. Of the three, free trisomy is by far the most common.
 * Appreciate that the field of clinical cytogenetics is dynamic, with evolving molecular tests, such as array comparative genomic hybridization (aCGH), that challenge genetic diagnosis and medical care
 * **aCGH** : slide or silicon matrix in which gene-specific probes are arrayed, covering the entire genome. Label patient DNA one color, control DNA another color; look for color variations showing deletion or duplication of genes.
 * Essentially looking at copy number of different genes.
 * Notice it can't detect balanced translocations of genes and it's not good at detecting mosaicism.
 * Notice that array studies are always confirmed with FISH.

=Chromosomal Abnormalities II and Imprinting=
 * Describe the mechanism of common chromosome structural rearrangements.
 * Common mechanism: __double-strand breakage__ in DNA and subsequent repair by __non-homologous end joining__ (which, recall, is kind of like having two blindfolded monkeys assemble a radio with glue: bits are always lost, put in backwards, eaten, etc).
 * Another common mechanism: __crossing-over between repetitive DNA sequences__. This can delete segments of a repetitive stretch, can delete on one stand and duplicate on the other, invert, reciprocally translocate, etc.
 * Compare and contrast various forms of balanced and unbalanced chromosomal structural rearrangements and understand their reproductive consequences.
 * **Balanced** : normal, but rearranged, complement of chromosomal material. Often phenotypically neutral.
 * Inversion (double-stranded segment flipped around, 5'-3' to 3'-5'):
 * **Paracentric** inversions exclude the centromere
 * **Pericentric** inversions include the centromere (inverted segment goes from //p// to //q// )
 * During meiosis, the chromosome now has to loop around to pair effectively with its homologue, which introduces a whole new set of problems with crossing-over. See PP slides if you're interested.
 * __Reciprocal translocation__ (breakage/reforming create recombination of two non-homologous chromosomes: effectively chromosome A gets a chunk of chromosome B and vice versa)
 * Notice this is different from just straight up translocation, which is when one chromosome sticks itself whole cloth right onto another one.
 * Observed in ~ 1:500.
 * This really messes things up during recombination in meiosis: creates "quadravalance" in which four homologous chromosomes align instead of two.
 * This can lead to disease states: chronic myelogenous leukemia from translocation of chromosomes 9-22.
 * Risk of lethality with reciprocal translocation ~ 5-10%.
 * __Robertsonian translocations__ (fusion event between long arms of two acrocentric chromosomes- means you lose stalks/satellites of those chromosomes)
 * Notice that the chromosome count goes down by one.
 * Notice that even so, this is considered a "balanced" rearrangement.
 * Notice that Robertsonian translocations can result in DS (trisomy 21) without an abnormal chromosome number (two normal 21s, one fused on another chromosome).
 * Frequent occurrence of this: chr.14 - chr. 21 fusion. Notice this often results in trisomy 21 (gamete has the copy of chr. 14 with 21 stuck onto it as well as the normal copy of 21).
 * **Unbalanced** : abnormal chromosomal context. Often phenotypically abnormal.
 * Deletion:
 * Flavor one: the deleted segment on one chromosome arm: produces a deleted fragment (not stably transmissible) and the transmissible rest of the chromosome. This is called a "terminal deletion."
 * Flavor two: the deleted segment contains the centromere (ie goes from one arm to the other). This is called an "interstitial deletion." Notice that the deleted segment can form into a ring chromosome (see below).
 * Duplication:
 * Generally less harmful than deletion
 * Isochromosomes (one in which one arm is missing and the other has mirrored itself, replacing [or displacing] the missing arm):
 * Most commonly found on the X chromosome
 * Sometimes found on chromosome 21:
 * **100% of the viable offspring of a carrier of isochromosome 21 are abnormal.** --since the progeny either have three 21 chromosomes or one (thus not viable).
 * Marker (ring) chromosomes:
 * Occurs when an interstitial deletion fragment becomes circular and (since it contains the centromere) is stably transmissible to offspring.
 * Recall that the majority of Down's Syndrome patients have trisomy 21 due to a maternal meiosis I nondisjunction event.
 * Assess the recurrence risks of various chromosomal rearrangements for the progeny of carriers of these rearrangements.
 * Most chromosomal abnormalities are unlikely to recur (caused by random event).
 * Some stable, balanced rearrangements in the parents (like some translocations of chromosomes) are transmissible to offspring.
 * Define and give examples of contiguous gene syndromes.
 * A disorder due to overexpression or deletion of multiple gene loci that are adjacent to one another.
 * Ex: Velocardiofacial syndrome (del 22q11) and DiGeorge syndrome (del 22q11)
 * Define the term "epigenetics" and understand how DNA and chromatin modifications may affect gene expression.
 * **Epigenetics** : Heritable changes in gene expression that occur without a change in DNA sequence.
 * Examples are patterns of reversible post-translational modifications of histones and pattern of DNA methylation (alters configuration of DNA, not DNA sequence itself).
 * Describe genetic imprinting and its molecular basis.
 * Small subset of genes that are inherited in a transcriptionally active state from one parent and transcriptionally inactive state from the other parent: this is called **genetic imprinting**.
 * Clinical interpretation: we're normally hemizygous (only one working copy of a gene) for all of our imprinted genes. This is synonymous with saying we're particularly vulnerable at all of those genes, since there's no backup.
 * Activation or inactivation seems to depend on the methylation or nonmethylation of cytosine residues in CpG islands in promoter regions of particular genes.
 * Methylation binding proteins attract other proteins that act as HDACs to silence transcription and compact chromatin at the site. (Point: generally, __DNA methylation leads to transcriptional inactivation__.)
 * Describe the different fates of DNA methylation imprinting patterns in germ line and somatic cells.
 * 3 rules for epigenetic DNA methylation:
 * Modification must be established during gamete genesis (all paternal gametes must be imprinted the same way, all maternal gametes must be imprinted the other way).
 * Modification must be stably maintained in somatic cells (which will contain half paternal and half maternal modifications) after fertilization.
 * Modifications must be reversible so that they can be reset during gametogenesis to transmit the appropriate sex-specific imprint to progeny.
 * Somatic cells: DNA methylation patterns (silencing/expression of imprints) are set and don't change (some genes are maternally imprinted, some are paternally imprinted).
 * In primordial germ cells, all alleles undergo universal demethylation (erasing the inherited imprinting pattern). Then they're methylated again in a sex-specific manner (if they're in a female, maternally methylated; if in a male, paternally methylated)
 * This happens to ensure that all genes coming from the father to offspring have ONLY paternally imprinted gene patterning, and that all genes coming from the mother have ONLY maternally imprinted gene patterning.
 * Describe the relevance of genetic imprinting in Prader-Willi and Angelman syndromes.
 * Both PW and Angelman syndromes have to do with a deletion in a region of chromosome 15.
 * This region codes for two imprinted regions (one per disease)- one is maternally activated (Angelman syndrome), the other is paternally activated (P-W syndrome).
 * So you have two copies of chr. 15, one from your mother, one from your father. The one from your dad has active Prader-Willi genes and inactive Angelman genes. The one from your mom has active Angelman genes and inactive Prader-Willi genes.
 * If the chromosome region with the PWS-active genes [paternal chromosome] is deleted, results in Prader-Willi syndrome.
 * Notice that this also results in the deletion of a Angelman gene region, but since it's an inactive region, no Angelman phenotype results.
 * This happens because the PW region on the other, maternal chromosome is inactivated due to imprinting-- thus no working copies of this region remain once the paternal genes have been deleted.
 * If the chromosome region with the Angelman-active genes [maternal chromosome] is deleted, results in Angelman Syndrome.
 * Notice that this also results in the deletion of a PW gene region, but since it's an inactive region, no PW phenotype results.
 * This happens because the Angelman region on the other, paternal chromosome is inactivated due to imprinting-- thus no working copies of this region remain once the maternal genes have been deleted.
 * One can think of PW Syndrome as "paternal insufficiency" and Angelman Syndrome as "maternal insufficiency."
 * Notice that both of these can occur in both male and female offspring. (imprinting pattern is dependent on your parents, not your gender.)
 * Describe the origin and clinical effects of uniparental disomies with regard to Prader-Willi and Angelman syndromes.
 * Here, disomy is when one gamete has two copies of a chromosome.
 * If that gamete fuses with another, normal gamete, the zygote will have trisomy for that chromosome.
 * If the zygote has a nondisjunction mitotic event early in life, may continue on with normal chromosome number, but has //both// chromosomes from one parent.
 * This means that whatever genes are meant to be activated from the other parent aren't activated.
 * This means if it's maternal disomy for chromosome 15 (no copies of chr. 15 exist from the father), it results in Prader-Willi syndrome.
 * Or if it's paternal disomy for chromosome 15 (no copies of chr. 15 exist from the mother), it results in Angelman syndrome.
 * Notice the frequencies:
 * PW: 70% occurs from a deletion in the paternal gene, 25% from uniparental disomy from the mother.
 * Angelman: 70% occurs from a deletion in the maternal gene, less than 5% from uniparental disomy from the father.
 * Uniparental disomy in the father is rarer since nondisjunction meiotic events in male gametes are rarer than in the female.

=Sex Determination=
 * Understand the biological advantages of sexual reproduction
 * Diploidy: protects against effects of mutation (still one working copy)
 * Recombination: creates new combinations of haploid genes in individual's germ line
 * Sex: allows random chromosomal assortment by combination of haploid cells
 * Sex also makes possible gender-dependent epigenetic imprinting.
 * This permits rapid evolution and increases survival of species through increasing genetic variability
 * Sexual dimorphism allows for division of labor and cooperation (not bloody likely).
 * Describe X chromosomal inactivation and its implications
 * All diploid somatic cells have a single active copy of the X chromosome, whether it belongs to a male or a female.
 * In normal females, this means one X chromosome is inactivated in any given cell. The other remains as a "Barr body."
 * Note that this inactivation is a **mosaic** : that is, there's a 50-50 chance of either X chromosome in a female cell being active or inactive.
 * How inactivation works: DNA methylation and modifications of histones.
 * How that works: XIST region on inactive X transcribes a special type of RNA that spreads across that X chromosome to coat it, modifying the genomic structure to attract DNA methylators and histone deacetylases (HDACs).
 * Note that not all genes on an "inactive" X chromosome are actually silenced; some of the genes (10-15%) in Barr bodies are still transcribed.
 * "Nonrandom X inactivation" occurs when an X chromosome is abnormal. This results in the abnormal X chromosome being preferentially inactivated. Instead of a mosaic pattern where a 50-50 random pattern of inactivation, you get almost all cells with an inactive abnormal X due to nonviability of cells with active, abnormal X chromosomes.
 * Can still have abnormal phenotypes due to translocations, etc, but rare.
 * Describe the genetic regulation of sexual differentiation
 * Before you get to that point, you need the **WT** gene, which directs the differentiation of the embryological genital ridge (which you need to have any gonads at all).
 * Gonadal differentiation is dependent largely on whether or not genes promoting the development of testes are present.
 * Specifically, it depends on the **SRY** gene on the Y chromosome, which drives differentiation of gonadal tissue into testes (without SRY, ovaries develop).
 * Other important genes for proper sexual differentiation: **SOX9** (interacts with SRY), **SF1**, **DAX1**
 * Mullerian inhibiting factor (**MIF** ) produced by testicular cells: allows paramesonephric ducts to atrophy away and the mesonephric ducts to grow.
 * Describe the basic embryology of dimorphic human reproductive organs
 * Testosterone (derived from testicular non-germ cells) drives the differentiation of external genitalia.
 * However, testosterone production largely driven by pituitary gland-- so problems with the pituitary gland can result in external sexual ambiguity.
 * Mullerian inhibiting factor, as mentioned, allows the formation of ductus deferens, etc, from mesonephric ducts. Notice that this is not dependent on the pituitary gland.
 * Describe the clinical characteristics of disorders of sex chromosomes
 * True hermaphroditism: 46 XX/46 XY: show ovaries, testes, partial formation of uterus
 * Sex reversals:
 * XX males in which the Y chromosome has translocated autosomally.
 * XY females in which regions of the Y chromosome have been deleted or certain sex-developing genes on the X chromosome have been duplicated.
 * 45 X : called **Turner syndrome** :
 * Normal early female gonadal development in utero, but degeneration of developing ovaries later in fetal life.
 * Clinical features: short height, perceptual disorders, coarctation of the aorta (narrowing of aorta between upper-body and lower-body supply), fused kidneys.
 * 47 XXY : called **Klinefelter syndrome** . Develop as anatomic males, but have degeneration of gonads. Infertile, low levels of testosterone development.
 * Clinical features: tall stature, gynecomastia (development of breasts).
 * Turner and Klinefelter are mostly driven by meiotic nondisjunction events.
 * Androgen insensitivity: presents as non-menstruating females.
 * Notice that androgen insensitivity does not result in a uterus (testes still produce working mullerian inhibiting factor even if the testosterone isn't able to affect development).
 * "Pseudohermaphroditism": Individuals with ambiguous external genitalia but normal ovaries or testes (not both).
 * Describe the clinical approach to disorders of sexual differentiation
 * Not much discussed in lecture or in the notes.

=Multifactorial Inheritance=
 * **Identify and describe the characteristics of diseases and other traits that demonstrate multifactorial inheritance**
 * Spectrum of disease: simple Mendelian to extremely complex multifactorial
 * Many diseases have characteristics that aren't explained by the genotype at the causative locus. In addition, different alleles at the same gene can result in different levels of severity (as in cystic fibrosis).
 * Multifactorial: combination of genetic variants and nongenetic factors.
 * Tend to aggregate in families but don't follow simple modes of inheritance.
 * **Specific characteristics of complex traits:**
 * **Incomplete** penetrance : not everyone with predisposing genetic variant develops the disease.
 * Ie: Type I diabetes (20% population have predisposing genotype, incidence is 0.4%)
 * **Variable expressivity**: No people with the same genetic variant have the exact same disease characteristics.
 * Ie: Maturity Onset Diabetes
 * **Heterogeneity** : "Same" or similar diseases can be caused by (1) different alleles at one location in the gene or by (2) alleles at different locations (or loci) in one gene or among many genes. **
 * (1) = allelic heterogeneity
 * (2) = locus heterogeneity
 * eg: cystic fibrosis (allelic: lots of alleles lead to CF, variable severity)
 * eg: Alzheimer's (locus: mutations in chr. 1, 14, 21 all lead to AD)


 * Phenocopies: People who have the disease (same clinical presentation) for reasons that aren't primarily genetic.
 * eg: thalidomide-induced limb malformation v. genetically-induced **
 * Give specific examples of diseases and other traits that demonstrate multifactorial inheritance
 * Some cancers
 * Diabetes, I and II
 * Alzheimer's
 * Inflammatory bowel disease
 * Asthma
 * Schizophrenia
 * Hypertension
 * Cleft lip/palate
 * Rheumatoid arthritis
 * Describe the strategies used to determine the relative importance of genetic vs. non-genetic factors in contributing to the variation in a complex trait
 * Epidemiologic twin, adoption, and immigration studies
 * Twins: compare monozygotic to dizygotic twins
 * Adoptive: compare biological siblings raised apart and adoptive siblings
 * Examine disease frequency and risk pattern in relatives
 * Notice that if you have one major gene associated with high penetrance of a given disease trait, that's about as simple-Mendelian as you get.
 * Understand the potential difficulties associated with quantifying the role of genetic factors in contributing to risk of disease at both the population level and the individual level
 * Study of heritability (heritability: proportion of total variance in a trait that's due to // genetic // variation).
 * High heritability: differences among individuals with trait can be largely attributed to differences in genetic makeup.
 * Low heritability: differences among individuals with trait can be largely attributed to differences in environmental factors.
 * Notice that high or low heritability can still mean that the other variability source is significant-- it's just not __as__ significant.
 * Re this bullet point: the emphasis here is that the roles of genetic and non-genetic factors vary from trait to trait and individual to individual-- thus hard to lay down strict guidelines for what markers mean what about disease predispositions.

=Autosomal Recessive Disorders=
 * locus: physical position of a gene on a chromosome.
 * alleles: different isoforms of same gene
 * Overview of Autosomal Recessive Disorders
 * (a) Know the common characteristics of disorders that are of autosomal recessive inheritance
 * Phenotype expressed only in homozygotes.
 * Males and females usually equally affected.
 * Horizontal inheritance pattern (sib-sib more than parent-child).
 * Parents of an affected child are obligate carriers (or affected themselves).
 * The recurrence risk for each potential child of two carriers is 1/4.
 * The chance of unaffected children of two carriers being carriers is 2/3 (unaffected means it's not the 1/4 that's homozygous recessive, thus 3 possibilities left, 2 of which are carriers).
 * Predisposition in certain ethnic groups
 * (b) Calculate allele frequency and carrier frequency of a given autosomal recessive disease when provided with the disease frequency, and vice versa
 * Frequency of disease = // q // 2. Can take the square root of that to get the allele frequency // q //, then subtract // q // from 1 to get the frequency of the wild-type // p // allele. Once you have // p // and // q // , you can calculate the carrier frequency by 2 // pq //.
 * If given // q //, can get // p // by subtracting // q // from 1; then you have enough information to get // q // 2 and 2 // pq // (thus calculate the carrier frequency and allele frequencies.
 * Notice that if all you're given is 2 // pq //, you don't have enough information to calculate anything else.
 * (c) Understand the following concepts:
 * 1) **Allelic heterogeneity** : Existence of multiple alleles of a single gene
 * 2) **Compound heterozygote** : A person who carries different mutant alleles at the same genetic locus (see "Biochemical Basis of Genetic Disease").
 * 3) **Parental consanguinity** : Parents sharing one or more common ancestors. (Obviously, we're all related if you go back far enough-- Wiki says consanguinity is usually defined as second-cousins or more closely related.
 * 4) **High-risk group** : An ethnic group in which an autosomal recessive disease occurs with higher frequency. This is high-risk both because of the increased frequency of the allele and also because people who intermarry within that ethnic group are at a much higher risk for being homozygous recessive for that allele.
 * 2) **Phenylketonuria** (PKU)
 * (a) Discuss the biochemical deficiencies in PKU patients and the appropriate treatments:
 * __PKU__: defect of phenylalanine (F) metabolism (can't break down F). Results in high F levels in the blood and high levels of F metabolites in the urine. Shows as hyperactivity, epilepsy, and mental retardation.
 * This is usually a result of a defect in the gene coding for phenylalanine hydroxylase (PAH), which converts F into tyrosine (Y).
 * Occasionally, it's instead a result of coenzyme genes, so that the reaction doesn't go forward despite having intact PAH enzymes.
 * Has a large variety of mutant alleles-- thus incidence of compound heterozygosity is fairly high.
 * Preventable: treatment with low-F diet prevents mental retardation. Notice that F is an essential AA and can't be eliminated from the diet completely. The diet can be discontinued after the school years, unless the patient gets, or plans to get, pregnant (see next).
 * (b) Explain maternal PKU and its treatment:
 * Pregnant women who have PKU and are not on F-restricted diets have a high incidence of miscarriage and developmental abnormalities in their children.
 * These abnormalities in children are not caused by the genotype of the child but the high levels of F in the maternal circulation when the child is // in utero //.
 * The developmental abnormalities are called **maternal PKU** since they aren't a result of the child's genes.
 * (c) Know newborn screening procedures for PKU and importance of the timing of the test:
 * Can be tested with mass spectrometry to look for abnormally high levels of F (as well as low levels of Y, its metabolite) in the blood.
 * Timing of the test is important: before ingestion of food, the new baby won't have elevated F/Y ratios. So take a baseline blood sample at birth, then wait for a couple of days and test again to compare.
 * **Alpha1-Antitrypsin Deficiency (ATD) **
 * (a) Know the clinical features of alpha1-antitrypsin deficiency and the influence of environmental factors on the expression and severity of the disease (ecogenetics):
 * Disorder is late-onset, more common among Northern Europeans.
 * 20-fold increased risk of developing emphysema; also much higher risk of developing liver cirrhosis.
 * Notice that smoking aggravates both problems ("environmental factors").
 * What's missing: the alpha1-antitrypsin enzyme.
 * (b) Which enzyme is the primary target of alpha1-antitrypsin?
 * Mainly targets and inhibits elastase, a serum protease, by irreversible binding.
 * Elastase is released by neutrophils in the lung to break down elastin (structural protein in the lung alveoli). Basically, elastase breaks down lung tissue to be remodeled.
 * If elastase isn't inhibited by alpha1-antitrypsin, lung tissue breaks down more quickly, causing inflammatory responses that cause the lung tissue to break down even faster-- eventually causing emphysema.
 * (c) Know the two most common mutant alleles that cause ATD and the severity of different allelic combinations. Why do some ATD patients have liver failure?
 * Not a lot of different mutant alleles:
 * Z allele: most severe and common form (ZZ genotype = 15% normal alpha1-antitrypsin function). Makes an improperly folded protein which gets stuck in the liver cells, which is why elevated rates of liver cirrhosis are observed in ATD patients.
 * S allele: less severe; makes unstable ATD proteins as opposed to stable but misfolded proteins (thus no liver disease).
 * Notice that ZZ smokers have an average life span of about 40 years. (ZZ non-smokers live an average of 60 years.)
 * **Tay-Sach Disease** (T-S)
 * (a) Explain the biochemical defects in Tay-Sachs disease and why the brain is the major target:
 * Early-onset, fatal disease targeting the CNS. Born normal, symptoms show about 9-12 months, usually death occurs 2-4 years.
 * The problem in patients is a defect in their ability to get rid of a ganglioside (lipid) that makes up about 5% of brain mass. Instead of being able to break it down and turn it over, the ganglioside gets stuffed into lysosomes until the lysosomes are massively engorged.
 * (b) Compare the biochemical and genetic differences between Tay-Sachs disease and Sandhoff disease:
 * T-S: mutant alpha subunit of the enzyme required to break down ganglioside
 * Sandhoff: mutant beta subunit of the enzyme required to break down ganglioside (also results in inability to break down other lipids).
 * (c) Know the high-risk group for Tay-Sachs disease and the available methods for carrier screening and prenatal screening in the high-risk population
 * High-risk: Ashkenazi Jews for the most part.
 * Notice that three mutant alleles account for > 95% of the mutations in that population; DNA testing for these alleles are offered for carrier and prenatal screening.

=Molecular Genetics of Hemoglobinopathies=
 * Describe the layout of the alpha- and beta-globin gene clusters and the switch between different forms of hemoglobin (Hb) during development. Explain the function of the locus control region (LCR).
 * All of the alpha genes are in the alpha-cluster of chromosome 16; all of the beta genes are in the beta-cluster of chromosome 11. Recall that hemoglobin is a tetramer, composed of two alpha and two beta subunits. (Also can consider it two alpha-beta dimers.)
 * Notice alpha and beta subunits have very similar sequences, structures, and heme-binding groups. That said, 2 alphas and 2 betas are vastly more efficient at binding oxygen than 4 alphas or 4 betas.
 * Alpha-cluster has this sequence : zeta-alpha1-alpha2.
 * Beta-cluster has this sequence : epsilon-gammaG-gammaA-delta-beta
 * (simplified) Normal development of types of globins: first zeta-epsilon (early embryo), then alpha-gammas (fetus), and finally alpha-beta (post-birth).
 * Cool thing: genes in cluster are arranged 5' to 3' in the order in which they will be expressed during development. This is called "globin-switching."
 * Major form of hemoglobin: A2B2. Minor form: A2D2. Delta globin is a minor auxiliary of beta globin.
 * Alpha replaces zeta and remains on throughout life.
 * Major form of fetal hemoglobin: A2G2.
 * Gamma is dominant beta cluster hemoglobin until birth, at which point beta form predominates.
 * The location of erythrogenesis changes as well: yolk sac to liver to spleen in utero, then bone marrow after birth.
 * Locus control region (LCR): 10-20 kbp sequence far upstream of the cluster.
 * In the globin clusters, each genes has its own promoter; LCR controls which of these promoters are turned on or off.
 * The LCR controls both the __timing__ and the __level__ of expression.
 * Most hemoglobinopathies are either __structural__ (altered globin properties), __thalassemias__ (low levels of synthesis of one globin), or __defective globin switching__ (hereditary persistence of fetal hemoglobin-- gamma hemoglobin continues at elevated levels through life).
 * Describe the mutations that cause sickle cell anemia and hemoglobin C disease and their consequences. Know the DNA diagnosis method of the sickle cell disease mutant allele.
 * **Sickle-cell anemia** : Affects __beta__-globin gene; caused by mutation in exon 1: A-to-T mutation at codon 6 in that exon causes glutamate residue to be replaced with valine. This makes it less soluble and more likely to polymerize, causing sickle-shaped agglomerations of globins to result, which then block up capillary vessels.
 * Usually heterozygotes for sickle-cell are phenotypically normal unless put through heavy exertion. These heterozygotes are said to have "__sickle cell trait__."
 * **Hemoglobin C** : Also an A-to-T mutation in codon 6 in exon 1 of beta-globin gene at a slightly different position. Changes the same glutamate residue to lysine, making the protein less soluble (though not as much as in sickle-cell). Less severe than sickle cell.
 * DNA diagnosis: use a restriction enzyme that cleaves at the normal site sequence in exon 6. With the mutation for sickle cell, can't cleave there and have a larger fragment in that position. Can use a DNA probe to detect these particular fragments and run PCR to detect larger or smaller amounts of cleavage at this site.
 * Know the **six** possible genotypes of alpha-globin locus, their clinical phenotypes, and the geographical distributions of alpha-thal-1 (--) and alpha-thal-2 (alpha -) alleles.
 * α -thal-1 (--): **caused by deletion of both copies of alpha globin gene in alpha cluster on the same chromosome.**
 * **(1)** Homozygotes (--/--): results in stillbirth.
 * **(2)** Heterozygotes ( αα /--): Mild anemia ("//alpha-thalassemia-1 trait// ").
 * Mainly found in __East Asian__ areas.
 * α -thal-2 ( α -): **deletion of one copy of alpha globin genes. 50% decrease in α-globin synthesis.**
 * (3)** Homozygotes ( α -/ α -):** Mild anemia in ("//alpha-thalassemia-2 trait// ").
 * (4)** Heterozygotes ( αα / α -): no disease phenotype.**
 * Mainly found in __African and Mediterranean__ regions.
 * ** α -thal-1/ α -thal-2 ( α -/--):**
 * (5) ** Compound heterozygotes, who have only 25% of normal alpha globin levels. Severe anemia, called "//HbH disease// ".**
 * Mainly found in people who have at least part SE Asian ancestry.
 * **(6)** Sixth** genotype is normal people, who are fine. **
 * Understand the following concepts about beta-thalassemias :
 * [Something to keep in mind: since there's two alpha globin genes on each chromosome, you effectively get four chances to have at least one of them work. With beta globin genes, you only get one per chromosome, which means the odds are less in your favor.]
 * (a) thalassemia major & thalassemia minor:
 * A clinical way of characterizing beta-thalassemias:
 * // Major // is characterized by severe anemia, in which most red blood cells are destroyed before being released into circulation.
 * // Minor // means a clinically normal carrier of one beta-thalassemia allele.
 * (b) beta0-thalassemia & beta+-thalassemia:
 * A molecular-biology/genetics way of characterizing beta-thalassemias:
 * beta0: Zero beta-globin synthesis. Caused by deletion of gene, nonsense mutation, frameshift mutation in early beta coding region. Generally this leads to death after birth.
 * beta+: Most common beta-thalassemia. Some beta hemoglobin still made. Often caused by mutations affecting transcription, protein stability, etc.
 * (c) beta0-thal allele & beta+-thal allele:
 * I really don't know what this is. I presume it has something to do with the beta0 and beta+ thalassemia characterizations, above-- maybe beta0 means that the allele will produce no function beta globin and beta+ means that it will produce a limited amount.
 * (d) simple beta-thalassemias & complex thalassemias :
 * A biochemical way of characterizing beta-thalassemias:
 * Simple: Caused by mutations or deletions that only target the beta globin gene.
 * Complex: Mutations or deletions that target not only the beta globin gene, but other genes in the beta cluster, or the LCR. Notice this can cause HPFH (below).
 * Explain hereditary persistence of fetal hemoglobin (HPFH) and its significance. Give examples of two types of mutations that are known to cause HPFH.
 * HPFH is a condition that results from not switching over to beta globin from gamma globin after birth (beta and delta globin genes deleted). As such, it has genetic components that are heritable. Two common genetic causes:
 * Large deletion that brings an enhancer closer to gamma gene. Leads to persistent expression of gamma hemoglobin in adult (enhancers overcome the repressors).
 * Point mutations in gamma gene destroy the targets of transcriptional repressors, which means the genes can't be turned off in adults.
 * Note that gamma hemoglobin can substitute for defective beta hemoglobins in part.

=Pharmacogenetics=
 * Know the difference between pharmacogenetics and pharmacogenomics.
 * Pharmacogenetics : Study of how variance in a single gene influences variability in drug response, usually based on prior knowledge of drug action pathways.
 * Pharmacogenomics : Study of how variance across multiple genes influences variability in drug response, usually not based on prior knowledge of drug action pathways.
 * As described in the notes, have a historical perspective on some of the key events that led to the establishment of pharmacogenetics as a discipline.
 * There were events. Some of them were key.
 * Know the cost, in terms of mortality, morbidity and health care dollars, of adverse drug responses in the human population.
 * Fourth to sixth leading cause of death in US hospitals.
 * Morbiditity/mortality estimated at $30 billion/year in US.
 * Understand the cost/benefit considerations related to widespread pharmacogenetic testing.
 * Benefit: reduce adverse drug reactions.
 * Cost: extremely expensive to genotype every person.
 * Know that drug metabolism has Phase I and Phase II steps and understand the characteristics of each as described in the notes.
 * __Phase I: "first pass" metabolism__: hydroxylates drug, usually by cytochrome P450 enzymes in the liver.
 * __Phase II: Conjugation reactions__: glycosylation or acetylation to deactivate drug, make it more soluble, and excrete it faster.
 * Know the list of known single gene differences and their known pharmacologic effects described in the notes.
 * Malignant hyperthermia:
 * Adverse response to __inhaled anesthetics__
 * Caused by defect in RYR1 and other __calcium channels__
 * Reaction to succinylcholine:
 * __Succinylcholine__ is a __paralytic agent__ for surgeries.
 * Reaction is caused by defect in __pseudocholinesterase__ enzyme.
 * Results in __prolonged apnea__ (suspension of breathing)
 * Glucuronyl conjugation:
 * Results in __bilirubin buildup__; caused by problems in __glucuronyltransferases__
 * Results in __Crigler-Najaar syndrome__
 * Isoniazide acetylation:
 * __Isoniazides__ are used as a treatment for __tuberculosis__.
 * Defect in __N-acetyltransferase-2__ (can't acetylate isoniazides).
 * Flushing response to alcohol
 * Caused by deficiency in __aldehyde dehydrogenase__.
 * Primaquine sensitivity
 * __Primaquine__: __anti-malarial treatment__.
 * Caused by defect in __glucose 6-phosphate dehydrogenase__ enzyme.
 * Cytochrome P-450 polymorphisms
 * Cytochrome P-450s are involved in metabolizing many different drugs.
 * Know that differences in drug response may be due to any of a number of different types of genomic variations.
 * Understand why it now appears that there may be more differences between human genomes than previously appreciated: know the types of variations (SPNs, structural variations, etc.) as described in the notes.
 * SNPs
 * Structural variations (duplications, insertions/deletions, inversions, etc)
 * Know why human genomic variation has been underestimated in the recent past.
 * Method used to map the human genome (shotgun sequencing) misses a lot of variation in highly similar sequences within the genome.
 * Know why pharmacogenomics is of great interest to pharmaceutical companies.
 * Reduce adverse drug effects
 * Target drugs to particular populations
 * MAKE A FUCKLOAD OF MONEY on account of they're the devil.
 * Understand what is known about variations in the CYP2D6 gene, e.g. what types of variations exist, what the range of responses are, and what types of drugs are involved, as described in the notes. Also know about the interethnic variation in CYP2D6 gene copy number and its consequences.
 * __CYP2D6__: enzyme responsible for ~20% of drug metabolism in the liver, across a wide variety of drug classes.
 * Over 75 variant alleles of this gene: SNPs, gene deletions, gene duplications.
 * Classification of patients based on phenotypes:
 * Ultrarapid metabolizers (UMs)
 * Extensive metabolizers (EMs)
 * Intermediate metabolizers (IMs)
 * Poor metabolizers (PMs)
 * Copy number variation: from 0 to 13 copies.
 * High copy number: Saudi Arabians and Ethiopians among others.
 * High copy number seems to correlate with rapid drug metabolism (less drug action, tapering out more quickly).
 * Know about TPMT variation and thiopurines and about CYP2C9 variation and warfarin.
 * TPMT: thiopurine-S-methyltransferase.
 * Involved in metabolism of thiopurines (used for cancer treatment).
 * Variation here seems to mainly be SNP-inactivation of the TPMT gene, which results in slower metabolism of thiopurine drugs (need smaller dose to be effective).
 * Zygosity of TPMT-inactivating mutations in patient indicates how much of normal thiopurine dose should be given.
 * CYP2C9:
 * Related to warfarin metabolism, as CRP2D6 is to thiopurine metabolism.
 * Inactivations in CYP2C9 are associated with lower rates of metabolism of warfarin (thus dosing requirements go down).
 * Understand how response to drugs can involve many different genes and pathways (not just drug metabolizing enzymes), as described in the notes on pharmacokinetics and pharmacodynamics.
 * (transporters, signal transduction downstream, etc.)
 * (and I thought he said several times in lecture that we __weren't__ responsible for the table in the notes.)
 * Understand the clinical relevance of drug target pharmacogenomics and the targets that are listed in the table in the notes.
 * Effectively not much different from "why drug companies want to use it," above.
 * Understand the "double-edged sword" nature of human genome variation.
 * Produces greater "insurance" against species- or community-wide catastrophes, and drives adaptive change, but also results in high disease incidence.

=Genetic Testing=
 * Be able to define what constitutes a ‘genetic test’:
 * (1) Analyzing an individual's genetic material to determine predisposition to a particular health condition or to confirm a diagnosis of genetic disease.
 * (2) Examining a sample of blood or other body tissue for biochemical, chromosomal, or genetic markers that indicate the presence of absence of genetic disease.
 * Notice this involves DNA and non-DNA factors, and looking at risk as well as disease.
 * Identify methods of ‘genetic testing’ that do NOT involve the direct analysis of DNA sequence
 * Biochemical tests (for amino acids, organic acids, etc), enzyme activity assays, protein electrophoresis, cholesterol testing, X-rays, medical history and family history, blood pressure, etc.
 * **Understand the basic approaches, advantages and limitations of the following types of genetic tests:**
 * Chromosome analysis**:**
 * Used when chromosomal abnormality is suspected. Often done with amniocentesis on pregnant women, or from peripheral blood.
 * **Method: stain, look at chromosomes under microscope, look for abnormal chromosome number and** large ** (3-5 Mbp) changes (large deletions, duplications, insertions, rearrangements). **
 * Note that it can't detect small chromosomal changes.
 * Fluorescent in situ hybridization (FISH)**:**
 * **Used to diagnose deletions, some translocations, abnormality of copy number (**smaller ** scale than chromosome abnormalities). Often used in prenatal settings and to detect certain cancer genes. **
 * Method: use fluorescently labeled DNA probes to pick up copies of complementary DNA in patient's denatured genetic material.
 * Note that FISH needs a known genetic sequence to work-- it can't detect unknown genes (it needs to be able to bind to a complementary sequence, which means that you need to know that sequence when you're making it). Ironically, you can't go 'fishing' for an unknown deletion with this method.
 * Restriction digests**:
 * Used when a known mutation alters the pattern of an endonuclease digest.
 * Method: use a restriction endonuclease and look at the sizes of the fragments that come out of it. If the mutation changes the normal fragmentation pattern, should show up on a gel or however else you want to detect it.
 * Note that this is pretty much useless unless a specific mutation occurs right at the restriction site of a given endonuclease.

Linkage analysis:
 * Linkage: the tendency for alleles physically close to each other on a chromosomal segment to be transmitted together, as an intact unit, through meiosis (not following Mendel's Law of Independent Assortment).
 * Can be used to determine the probability of an offspring inheriting a DNA locus containing a mutation, based on looking at some nearby or 'linked' allele that presumably is inherited along with the disease allele.
 * You look for a pattern of alleles that indicates disease by tracking the disease through the family, then test for that pattern in patient.
 * Notice that this analysis doesn't identify the particular mutation, just that one's occurred.
 * Genetic sequencing:
 * Amplify given sequence of DNA with PCR; purify. Use radiolabeled oligonucleotides to replicate DNA (see 10/26/07, "Tools of Molecular Biology") to determine the sequence of a given gene in a given individual.
 * Notice this can miss mutations in promoters, some introns, etc.
 * No detailed questions on exam.
 * Detection of tri-nucleotide repeats:
 * Can use Southern blotting: depending on how many repeats are present in the DNA, it's run slower on the gel. Generally, the slower the DNA runs on the gel, the worse the phenotype is.
 * Microarrays:
 * Can test the //presence// or the //activity// of genes (which are being transcribed, which aren't). Can study transcription of 1000's of genes at once using microarrays.
 * Effectively you pick up cellular material (containing DNA/mRNA) from the patient, purify it, and "dot" it across a glass slide. On that glass slide are lots of different DNA probes, one for each gene you want to look at. Once you "dot" the same amount of cellular mRNA onto each such DNA sequence and allow the probe to hybridize, you can detect whether mRNA for that gene is being expressed, and at what level, in the patient's cells.
 * No detailed questions on exam.
 * Interpret genetic testing results and distinguish between ‘informative’ and ‘non-informative’ results.
 * __Informative__: The genetic test definitively proves or disproves presence of disease.
 * __Non-informative__: It's possible to have false negative results and false positive results.
 * Explain how allelic heterogeneity and genetic heterogeneity can affect the sensitivity of genetic tests.
 * Allelic heterogenuity: multiple mutations in a given gene can cause disease (thus have to test for all of them to be sure).
 * Genetic heterogenuity: mutations in multiple genes can cause disease (again, have to test them all to be sure).
 * So with sickle-cell, it's one gene, one mutation: if you don't have the mutation, you don't have sickle-cell. On the other hand, cystic fibrosis shows up on one gene but 1000's of mutations-- so testing for one or two is non-informative.

=Treatment of Genetic Diseases=
 * Understand the concept that while curing genetic diseases remains challenging, many genetic diseases are amenable to some level of treatment/management.
 * Can be either medication-based or behavioral (diet, etc).
 * Recognize some of the genetic conditions that currently can be treated and those for which treatment may soon be available.
 * __Disease__
 * __Intervention__
 * __Substance/Technique__
 * G6PD deficiency
 * Avoidance
 * Antimalarials, barbituates
 * PKU
 * Diet Restriction
 * Phenylalanine-depleted diet
 * Galactosemia
 * Diet Restriction
 * Galactose-depleted diet
 * Hypothyroidism
 * Replacement
 * Thyroxine
 * Biotinase deficiency
 * Replacement
 * Biotin
 * Urea cycle defic.
 * Diversion
 * Sodium benzoate
 * Hypercholemia
 * Diversion
 * Oral resins
 * Hypercholemia
 * Inhibition
 * Statins
 * Hyperchol.emia
 * Depletion
 * LDL apheresis
 * (Notice variety of interventions above: avoidance, diet restriction, substance replacement, diversion to a different chemical pathway, inhibition of pathways, depletion of overexpressed substances)
 * __Strategy__
 * __Example__
 * __Status__
 * Cofactor admin.
 * Biotinase defic.
 * 50% patients responsive
 * Replace extracellular protein
 * Factor VIII in hemophiilia, α1-antitrypsin
 * Effective
 * Replace intracellular protein
 * ADA deficiency
 * Effective
 * Targeted replacement of intracellular protein
 * Gaucher disease
 * Effective
 * Identify examples of pharmacogenetic management of patients (e.g. medication selection and dosing).
 * Mentions cytochrome P450 in the notes (recall, is responsible for most of first-pass metabolism in the liver) as well as N-acetyltransferase (recall, is responsible for metabolizing isoniazides). Ideally, should figure out if there's a defect in either of those enzymes before you go giving standard doses of the drugs that they metabolize.
 * Also mentioned in lecture:
 * Copy number of CYP2D6 gene: determines fast or slow metabolism of certain drugs.
 * TMPT mutations inactivate body's ability to metabolize thiopurines (thiopurines used to combat common cancers in children).
 * Be able to discuss examples of genetic disorders that are treated on the basis of protein replacement therapy.
 * Alpha-1 antitrypsin deficiency can be addressed by giving the patient recombinant AT1; if caught early, can prevent accumulation of elastase-mediated lung injury.
 * // Fabry disease // (deficient in alpha-galactosidase A enzyme, leading to buildup of galactosides in lysosomes): leads to kidney disease, neuropathy, and cardiac complications. Can be treated with recombinant alpha-galactosidase specifically targeted to the lysosome.
 * // Pompe disease // : enzyme missing (alpha-glucosidase) that breaks down glycogen, leading to muscular failure. Can be treated in a similar way with IV drugs.
 * Identify the principles that need to be addressed to ‘cure’ a genetic disease through gene therapy.**
 * The idea is to introduce DNA/RNA into human cells to cure/slow progression of the disease.
 * Considerations:
 * Easy production
 * Targeting: specificity
 * Sustained production
 * Efficacy
 * Integration
 * Size capacity
 * Toxicity
 * Retroviral (RNA viruses), Adenoviral (DNA viruses), Non-viral (insert DNA in lipids):
 * Retroviral therapy:
 * Advantages: integrate into host genome, minimal host immune response
 * Disadvantages: limited insert size, able to infect only dividing cells
 * Safety: risk of insertional mutagenesis/germline integration
 * Efficiency: Retroviral titers relatively low-- efficient at infection, but only in dividing cells.
 * Duration: The gene can be passed on to the daughter cells of the host.
 * Adenoviral therapy:

Advantages: wide variety of cell types able to be targeted, large insert size, stable


 * Disadvantages: doesn't integrate into host genome, transiently expressed, there's a risk of malignant transformation, can be severe immune response.
 * Safety: Lower risk of insertional mutagenesis, but note potential for severe immune response.
 * Efficiency: Can infect non-dividing cells; possibility of higher titers.
 * Duration: Typically short-lived (not passed to daughter cells).


 * **Non-viral therapy:**
 * Advantages: very large insert size (can deliver mini-chromosomes), minimal host immune response.
 * Disadvantage: low efficiency, transiently expressed.
 * Safety: Does not integrate into host genome.
 * Efficiency: Low due to degradation by cellular mechanisms.
 * Duration: Typically short-lived (not passed to daughter cells).


 * Understand the theoretical risks of gene therapy.
 * There are ethical risks to gene therapy (no kidding).
 * There are physical risks to gene therapy (no kidding).

=Finding Disease Genes= [Note that these are kind of sketchy, partly because I'm not sure how well I understand this and partly because the lecture and notes didn't always line up well with the LO's. Take with a larger than usual grain of salt.]
 * Understand the concept of individualized medicine and how this will change the traditional medical model:
 * Effectively can predict disease before it happens in the individual, as well as treating according to individual genotype.
 * Know what evidence is usually taken as evidence that a disease/trait involves a genetic component and how this is measured:
 * Honestly, I'm not sure what he means here. I would guess it probably involves looking at heredity patterns as a first step. He may be referring to this:
 * __Positional cloning__: make use of linkage analysis to determine a disease gene's position in genome, then identify gene(s) involved, using human genome project data.
 * Know the two principal types of polymorphic markers typically used in genomic medicine and the differences between them:
 * Notice that you can only track __differences__ between people or through families.
 * __Microsatellites__: tandem repeat segments to fingerprint individuals and establish framework against which to locate genes
 * Single-nucleotide polymorphisms: single-nucleotide differences between individuals.
 * Notice that frequency of SNPs vary between ethnic groups
 * Copy-number variations: not much known regarding their disease significance.
 * Understand **haplotypes** and haplotype blocks:
 * Haplotype: As I understand it, a haplotype is a pattern of polymorphisms over a region of the genome.
 * Haplotypes are differentiated from each other by recombination events.
 * International haplotype mapping project: determined allelic frequencies in various ethnic groups, how they fit together in genome
 * Haplotype blocks: patterns of polymorphisms (SNPs) that stay the same over long periods of time. These are generally clusters of genes on areas of the genome that don’t exhibit much recombination and thus stay fairly stable.


 * [How to find disease genes:]
 * __Hypothesis-driven__: think a given gene is responsible, sequence it (candidate gene sequencing), look for mutations in that gene. Most of the time, the hypothesis is incorrect, especially in diseases partially due to environmental factors or diseases due to a variety of genes.
 * Candidate gene __association__ studies: look for mutations in genes nearby or associated with gene of interest.
 * Notice that in hypothesis-driven research, can't discover new genes or pathways, just features or patterns among known genes.
 * Most of these studies are case-control studies.
 * Most of them are also completely wrong-- 96% false positive rate.
 * Other type of study: "family-based," compares allele transmission from parents to patients.
 * "__transmission disequilibrium test__": compares transmission frequency of marker alleles from parents to affected offspring (if gene causes disease, transmission should be higher than chance [50%]).
 * __Discovery-driven__: don't have a particular gene in mind before you start looking.
 * __Genetic linkage__ studies: look for regions of genome that are systematically co-inherited along with disease: can discover new genes within regions of genome that you mark as relevant to disease.
 * Obviously this is easier with Mendelian alleles that have strong phenotypic effects and are fairly uncommon.
 * __Genome-wide association__ studies: the handout's definition is wrong here. NIH defines these as "any study of genetic variation across the entire human genome that is designed to identify genetic associations with observable traits (such as blood pressure or weight), or the presence or absence of a disease or condition."
 * I gather that you're effectively doing case-control studies looking at not just a few genes of interest but thousands of polymorphisms across the entire genome. With haplotyping, I think you can define more precisely what genetic group a certain person belongs to, so you can match them more accurately with others from that genetic group as controls. Presumably this means that your false-positive rate falls dramatically and that you can accurately pinpoint the genetic basis for disease much more quickly.
 * Understand the differences between, appropriate applications, and limitations of:
 * A) Genetic linkage studies :
 * (i) Know how recombination can define genetic intervals:
 * Close-together genes on chromosome: less recombination, more often inherited together.
 * Far-apart genes: more recombination, less often inherited together.
 * (ii) Know LOD score criteria for linkage:
 * LOD is a statistical measure of likelihood that loci are linked together given the inheritance/disease pattern
 * (iii) Know definition of centiMorgan (cM):
 * One cM is 1% recombination between any two genetic loci per meiosis.
 * Used to measure "genetic distance" between two genes (or, inversely, "linkage" between two genes).
 * B) Genetic association studies :
 * (i) Gene-specific: (see above)
 * (a) Know strengths and weaknesses of case-control studies:
 * "controls" often are from a genetically distinct group from the cases. Also, looking at one gene may not give you a good picture of what's going on unless you get crazy lucky.
 * (b) Know strengths and weaknesses of family-based studies:
 * Strengths: can be fairly sure your controls are from the same genetic group as your cases. Weaknesses: limited external validity (thank you, biostats) and difficulty tracking everyone down and getting them in one place.
 * (ii) Genome-wide:
 * Test all parts of genomes simultaneously between individuals for patterns of SNPs (look for SNP patterns highly associated with disease); look for differences that are statistically significant.
 * Requires large number of cases and controls but not limited to families.
 * Works best for common alleles with strong or weak effect
 * Can locate mutations with high accuracy

=Genetic Counseling=
 * Identify the goals of genetic counseling.
 * comprehend medical facts, understand condition's heredity, understand options for dealing with recurrence risks, adjust to condition
 * Recognize the basic tenets and the ethical principals of genetic counseling.
 * educational, nondirective, unconditional, supportive
 * respect for patient autonomy, beneficence, nonmalfeasance, justice
 * Recognize indications for genetic evaluation and counseling.
 * Hereditary condition in family, fetus/child with birth defect, child with mental retardation, exposure to carcinogen, consanguinity, advanced maternal age, family history, ethnicity, etc.
 * Recognize reproductive options currently available for families at increased risks and apply appropriate options based on genetic condition and family.
 * Recognize factors that may impact the client's perception of risk, selected course of action, and utilization of services.
 * Honestly, most of these are pretty obvious.
 * Participants will gain an appreciation of emotional and ethical issues associated with presymptomatic testing for adult onset disorders lacking effective treatment therapies.

=Autosomal Dominant Disorders=
 * Know the characteristic features of autosomal dominant inheritance.
 * Disorder is expressed in the heterozygote.
 * Affected individuals have at least one affected parent, unless it's a new mutation.
 * Offspring of an affected parent have a 1/2 chance of inheriting the defective gene (and thus having the disorder).
 * Tends towards a "vertical pedigree": disorder appears in each generation, as opposed to the "horizontal pedigree" of autosomal recessive.
 * Autosomal (not sex-linked).
 * Frequently have late-onset (ie Huntington's disease).
 * Frequently involve structural protein defects (ie Osteogenesis imperfecta).
 * Understand why autosomal dominant disorders are in general less lethal than other inherited disorders, and why they show such a variable phenotype even within families.
 * If they were particularly lethal as well as dominant, they would have died out (not have had a chance to be transmitted).
 * This implies that AD diseases aren't magically less lethal than AR diseases-- just that the lethal AD mutations don't reproduce successfully (thus have no pedigree).
 * What factors may complicate the assessment of an autosomal dominant pedigree? What is the difference between penetrance and expressivity, and between genocopy and phenocopy when describing an inherited disorder.
 * AD diseases tend to have a wider range of clinical presentations than AR.
 * Penetrance tends to be more of an issue (whether or not a diseased individual shows the disease phenotype).
 * Expressivity as well (to what degree the affected individual shows the phenotype).
 * Genocopy: A mutation in a different gene causing the same syndrome.
 * Phenocopy: A syndrome caused by environmental factors that mimics a genetic disease.
 * Understand the characteristic features and molecular basis of:
 * Achondroplasia : Most common form of dwarfism. Results from a mutation in FGF-R3 protein, which leads to its ligand-independent activation, inhibiting chondrocyte synthesis (chondrocytes: cells causing long bone growth). About 80% of achondroplasia cases are new mutations.
 * Marfan Syndrome: Connective tissue disorder; musculoskeletal and cardiovascular (weakening of connective tissue in aorta) problems as well. Caused by mutation in fibrillin gene on chromosome 15. Physical exertion can cause dissection of the aorta.
 * Neurofibromatosis-I : Frequency 1/3500, many new mutation cases. Show café-au-lait spots and peripheral nerve tumors later in life. Caused by a mutation in the NF-1 gene on chromosome 17, which may be a tumor suppressor gene (turns off oncogene // ras // ).
 * Huntington's Disease : Frequency 1/10,000; usually late-onset in 3rd or 4th decade. Causes neuronal atrophy in the caudate nucleus of the basal ganglia (resulting in involuntary muscle twitching and jerking), slowly progressing to death in 5-15 years. Caused by expansion of CAG-repeat in HD gene on chromosome 4. Notice homozygosity for this disease is no different from heterozygosity in terms of clinical symptoms. Notice also that early-onset Huntington's tends to occur when it's inherited from their fathers (expansion of CAG repeat occurs during __male__ genetic transmission).
 * Familial Alzheimer Disease : Tends to show early-onset Alzheimer's. Notice that the great majority of Alzheimer's is sporadic.
 * What is a paternal age effect? What problems do late-onset disorders create for genetic counseling?
 * Paternal age effect: increased frequency of inheriting disease gene when father is over 39 years old.
 * Problems for counseling: since disease may not have shown by the time the parents want to have children, may not be able to counsel them accurately on the genetic risks posed to their offspring.

=X-Linked Recessive Inheritance and Mitochondrial Diseases=
 * Know the characteristic features of X-linked inheritance.
 * Incidence is primarily in males.
 * Trait can't be transmitted from father to son.
 * Disease gene is transmitted from an affected father to __all__ his daughters, who will either be carriers or show the trait depending on whether they also inherit a diseased X gene from their mother.
 * Carrier females transmit the gene to 1/2 of their sons and daughters.
 * Occasional clinical manifestation in carrier females.
 * Disorders are generally severe, with early onset.
 * If the disorder is lethal (or causes infertility) in males, 1/3 of the male cases are new mutations (mother not a carrier).
 * When do females show clinical manifestations of X-linked disorders? When do females show “full blown” X-linked recessive disease?
 * Lyonization (inactivation of one X chromosome in any given cell): sometimes the abnormal X is activated in cells in a given tissue.
 * Females can show full-blown X-linked recessives when both parents have the genes, or have Turner's syndrome (one X chromosome), or have a translocation of the affected portion of the X chromosome onto an autosomal chromosome.
 * Why are about 1/3rd of the individuals who inherit an X-linked recessive disorder that is eventually lethal, considered to be new mutations?
 * About 1/3 of inherited, diseased X chromosomes are in boys, who will die before reproducing. If the disease rate stays the same, that means that 1/3 of the disease states result from new mutations. (note this isn't exact but a good approximation.)
 * [Notice the following descriptions of intergenic and intragenic recombination: // intragenic // recombination results in a swapping of chromosomal material in the middle of a gene (thus makes a hybrid gene), // intergenic // recombination results in a swapping of chromosomal material between genes (thus no hybrid genes).
 * What are the clinical, biochemical/cytogenetic and molecular features of:
 * __Red/green colorblindness__: Affects ~8% of Causasian males; due to defective red or green pigmentation (opsin) gene on the X chromosome. Often results from intragenic (hybrid pigmentation) or intergenic (non-hybrid pigmentation) recombination between red and green genes-- frequently occurs due to misaligning at meiotic recombination, since red and green genes are very similar. Notice that a locus control region only allows the first two opsin genes to be expressed (thus if you have a red gene and a hybrid, mostly-red gene, even if you have a few more green genes after that, you're still green-colorblind).
 * __Hemophilia A__: Frequency is 1/10,000 male newborns in US, shows a defect in anti-hemophilia globulin (Factor VIII-- effectively, can't clot), often resulting from intragenic inversion on X chromosome. Notice that recombinant factor VIII can now be made and used to treat this disease.
 * That intragenic inversion often results from the long arm of X flipping back and recombining with itself at factor VIII site (results in particularly severe hem A).
 * Can diagnose affected or carrier status with restriction fragment length polymorphism, PCR.
 * Genocopies:
 * Hemophilia B: X-linked recessive disorder in Factor IX.
 * Von Willebrand's disease: AD-inherited disorder affecting Factor VIII.
 * __Duchenne muscular dystrophy__: Fatal disease, frequency 1/3,500. Involves progressive muscle weakness. Characterized by a defect in dystrophin protein production (dystrophin anchors the actin filaments to the muscle cell wall), usually caused by frameshift deletions in the extremely large DMD gene. Notice that there are less severe forms of the disease ( // Becker's MD // ) caused by in-frame deletions within the gene (Becker's is // allelic // with Duchenne's).
 * Notice that disease can sometimes be shown in heterozygote carriers when the affected X chromosomes translocated to autosomal chromosomes.
 * __Fragile-X syndrome__: Characterized by a constriction near the distal end at long arm of X chromosome. Results in moderate retardation with dysmorphic facial features. There is reduced penetrance in men (often don’t show symptoms).
 * Caused by an expansion in the CGG repeat region of the FMR1 gene, enhancing methylation and silencing FMR1.
 * Frequency is about 1/4000 male births (lecture says 1/1000-1/1500).
 * CGG triplet expansion occurs during __female__ genetic transmission.
 * [__Lesch-Nyhan disease__: very rare. Involves self-mutilation.]
 * X-linked dominant example: Vitamin-D resistant ricketts.
 * What are the characteristic features of Maternal Inheritance pedigrees? What genetic material is mutated? Clinical symptoms of these diseases are usually restricted to which tissues?
 * Diseases transmitted by a mother to all of her children, male and female.
 * Notice that affected males can't pass it on to any of their children.
 * Due to mitochondrial defects.
 * Diseases usually assoc. with oxidative phosphorylation and occur in tissues with high energy requirements-- brain, eyes, muscle, heart, kidneys, and liver.
 * Example: Leber's hereditary optic neuropathy.

=Research Ethics I= [Again: this is not really learning-objective friendly. Use your common sense here. It's not okay for parents to say "Sure, test new random drugs on my kid without any connection to therapy." It's not okay for researchers to promise potential subjects that the drug will cause peace and happiness in the Middle East, or to say "it's perfectly safe" while ducking behind lead-lined barriers (though my dentist still gets away with this). Non-PC way of saying all this: IRBs are there to cover institutions' collective asses by making sure nothing gets approved that could cause lawsuits to be filed against the institution.]
 * Identify the major responsibilities of Institutional Review Boards.
 * Apply the ethical principles that underlie the responsible conduct of human subjects research to three case studies.
 * Understand the role of the informed consent process in human subjects research.
 * Explore the significance of coercion and decision-making capacity to the informed consent process.
 * Identify some of the risks that financial conflicts of interest pose to the ethical conduct of human subjects research.

=Molecular Analysis of Mendelian Disorders=
 * What are restriction fragment length polymorphisms (RFLPs), and do they have to be within the gene in question to be informative? Are RFLPs ever 100% accurate?
 * DNA fragments generated by restriction endonucleases.
 * Use A: Since sometimes the genetic sequence at a restriction site mutates, this can make the site either more or less amenable to being cut. By comparing the restriction length fragments from a subject to a control, "normal" DNA sequence, you can tell if the sequence at that point has changed by looking at the size of the fragments that are generated.
 * Use B: Can use that principle, combined with observations about who's sick and who's not, to establish and follow linkage between a distant gene and the disease gene. The restriction sites don't have to be within the gene itself to be used to diagnose disease-- they just have to be within a gene that's linked to it (low recombination rate). Can only use this within families in which you can match the different restriction lengths to a phenotype.
 * Further note on this: effectively you're using a mutation on the same chromosome as a disease gene to 'follow' that portion of the chromosome through offspring. As long as the portion of the chromosome that you're following doesn't swap over to a different chromosome during meiosis, you can use the fragment length of an individual's DNA to predict whether or not they've inherited the chromosome that you know (from empirical evidence) causes the disease.
 * Generally this has about 90% accuracy (10% chance of recombination throwing off linkage).
 * Notice you don't need to know much of anything about the disease gene itself to follow it with this technique.
 * Know how synthetic oligonucleotide probes can provide specific information as to the inheritance of a particular mutation.
 * Once you know the sequence across a given possible mutation site, can make two different oligonucleotides, one with the normal and one with the disease sequence, and use hybridization to see which one binds to the patient's DNA.
 * Note can use this for alpha-1 antitrypsin deficiency (distinguish between the point mutation variations in alpha-ATD) and certain beta-thalassemias.
 * Understand how PCR technology has improved the speed and specificity of molecular diagnosis of specific genetic disorders.
 * Recall that PCR, given a 5' "priming" sequence, can massively replicate DNA very quickly.
 * If you use primers that are designed to either bind to a 'normal' DNA sequence at the disease site or to a specific disease DNA sequence, then PCR will either amplify tremendously or not at all depending on whether that sequence is present or not, and the difference in expression can easily be shown on a gel.
 * Specifically, can use to look for specific cystic fibrosis allele mutations.
 * Appreciate how DNA microarrays will greatly improve diagnosis of multifactorial genetic disorders.
 * Basically it's more efficient. You can look at thousands of different SNPs at once and establish a "constellation" or pattern with which to predict or diagnose disease states.
 * What is "genetic anticipation", and how does it relate to triplet repeat expansion disorders?
 * It's when the symptoms of a genetic disorder show up at an earlier and earlier age as the disease is passed from generation to generation.
 * Effectively, here's the thing. Say you have nine CAG repeats in front of some gene or other. Say that's enough to cause disease symptoms at age 60. Suppose, during meiosis in your germ cells, the chromosomes line up slightly incorrectly at this point on account of all the repeats, and suppose that a cross-over occurs in which, after the sixth CAG repeat on one chromosome, everything after the third CAG repeat on the other chromosome is transferred and vice versa. Now you have two recombinant chromosomes, one with 12 CAG repeats and one with 6. Anticipation, in this context, is the process in which more and more repeats are accumulated this way through generations.
 * When do triplet repeat expansions cause disease? What is the current thinking as to how trinucleotide repeats expand, and why their polyglutamine containing gene products lead to neuronal cell death in HD?
 * They cause disease in a variety of ways: they can cause a protein to be nonfunctional (ie methylation of CGG-rich region in Fragile-X syndrome), they can make an abnormal protein with novel function (ie Huntington's Disease, in which the abnormal huntingtin protein now interacts with a variety of transcriptional regulators), or they can make novel RNA that futzes about with things it shouldn't (ie myotonic dystrophy, in which the abnormal RNA binds to all kinds of receptors).
 * As far as how they expand, it occurs during replication, repair, or recombination:
 * During replication: may have something to do with hairpin loop formation (DNA bonding with itself) on lagging strand during Okazaki fragment synthesis.
 * During repair: may be associated with mismatch repair proteins putting in too many repeats, forming a DNA hairpin.
 * During recombination: may involve recombination within the repeating tract, particularly when the chromosomes aren't correctly aligned.
 * Polyglutamine-containing proteins: occur in Huntington's from CAG-repeats. Cause trouble both because they aggregate with each other in beta-sheets and also interact with a variety of transcriptional regulators abnormally.
 * Understand how to calculate recurrence risks for genetic diseases based on pedigree information and molecular analyses.
 * I believe this is the whole "mother's brother's dog's cousin has the disease, what's the risk to the mother's child" thing. Need to be able to distinguish autosomal recessive/dominant and X-linked recessive, also probably X-linked dominant and maternally inherited patterns. Remember the thing about 1/3 of all fatal X-linked recessive diseases being new mutations.

=Molecular Basis of Carcinogenesis I, II, III= [These are a little scattered, partly because it's new to me and partly because Dr. Sclafani is often more enthusiastic than he is organized.] [Notice that the notes have the following unelaborated steps: Tumor initiation, promotion, conversion, progression.]
 * Describe the properties of malignant cancer cells. You should be able to give at least five different properties.
 * Phenotype properties:
 * Unresponsive to normal signals for proliferation control.
 * De-differentiated (lack specialized structures of tissues in which they grow).
 * Invasive (can grow out into neighboring tissues).
 * Metastatic (can shed into circulation and proliferate elsewhere in the body).
 * Generally it's the metastasis that's fatal in cancers.
 * Clonal origin (derived from a single cell).
 * Increased transport of glucose.
 * Lack of contact inhibition (will grow over each other).
 * Immortality (ability to grow indefinitely).
 * Can grow without an attachment to a solid substrate.
 * What is the multi-step process for carcinogenesis? You should be able to discuss the relative importance of heredity and the environment and why early events may include mutations in DNA repair genes.
 * Not considered to be an inherited disease (not inherited as a single Mendelian gene).
 * On the other hand, cancer __susceptibility__ genes are certainly heritable (see Knudson below).
 * Carcinogenesis is characterized by the accumulation of many genetic alterations or mutations, particularly over a long period of time-- thus age is strongly associated with cancer, as are environmental factors that produce high rates of mutations.
 * If DNA repair genes are damaged early on, the rate at which you accumulate DNA mutations - since you can't repair them as well - goes up markedly.
 * Multi-step process for cancer:
 * (1) Normal cell
 * (2) Increased proliferation: With a mutation or two, an immortalized cell (see below for examples).
 * (3 + 4) Early/progressive neoplasia: With a few more mutations, get abnormal growth patterns.
 * (5) Carcinoma: A full-on tumor.
 * (6) Metastasis: A tumor that's spreading through the circulatory system.
 * I think the point he's trying to make here is that you need a fairly wide assortment of mutations (though order isn't important) to result in a cancer:
 * Turn on oncogene or make oncogene protein much more active
 * Turn off tumor suppressor genes (both cell cycle regulatory and DNA repair genes)
 * Turn off apoptotic genes and turn on anti-apoptotic genes.
 * What types of genes are usually mutated in tumor initiation? Describe the effect on cellular proliferation that the product of these genes has.
 * Either activated oncogenes (proliferation genes) or silenced tumor suppressor genes.
 * Oncogenes: accelerate proliferation. Tumor suppressors: slow down proliferation.
 * What type of cytogenetic abnormalities are associated with malignancy? You should be able to give at least two different examples.
 * Translocations of chromosomes, deletions on chromosomes:
 * Can activate oncogenes (for example, by putting an extremely active promoter upstream of one).
 * Can inactivate tumor suppressor genes (for example, by translocating another gene into the middle of the tumor suppressor sequence)
 * Notice that this is kind of a silly point to make, at least on its face. Anything that can bring a promoter nearby an oncogene (like pretty much any chromosomal rearrangement) or anything that can interrupt transcription or promotion of a tumor suppressor gene (likewise) can be associated with malignancy.
 * Notice that aneuploidy (recall: loss or gain of a chromosome) is associated with a poor outcome in many cancers.
 * What events can produce LOH? Give at least two examples and state how they support Knudson’s theory.
 * LOH = Loss Of Heterozygosity.
 * Means you inherit a heterozygous state (say, for a working tumor suppressor gene), but convert to (negative) homozygosity at some point.
 * Loss of this heterozygosity means you lose the one working copy of the gene that you have, through:
 * mutation
 * mitotic recombination
 * chromosome loss
 * and/or environmental factors
 * Knudson sez: If you're heterozygous, you have one "strike" against you through your genes (one copy of suppressor gene knocked out in your parents' passed-on DNA). If you have one more "strike" (ie, exposure to UV light causes an unrepaired mutation in the other copy of that gene), you're unable to produce that tumor suppressor gene product at all-- which leads, potentially, to cancer.
 * Example: Familial retinoblastoma vs acquired retinoblastoma (in children vs. adults, bilateral vs. unilateral)-- former is easier to acquire since it only requires one mutation event.
 * Are cancers associated with both dominant and recessive syndromes? You should be able to give a different example of each type.
 * Familial retinoblastoma: A recessive disease but inherited in a dominant way: need both tumor suppressor (RB) genes knocked out to show a phenotype (thus recessive), but everyone who inherits heterozygosity winds up with the disease (thus dominant) due to LOH problems.
 * Notice that this inheritance pattern shows a vertical pedigree (looks like autosomal dominant).
 * Sporadic retinoblastoma: Mutations in both tumor suppressor genes needed to knock out function- thus not strongly inherited (not sure how he's tying this to the LO, as somatic sporadic mutations shouldn't be inherited at all).
 * Describe how the RB (retinoblastoma) gene was first identified. You should be able to describe the important cytogenetic and molecular evidence.
 * Sorry, I was scrambling around trying to figure out the last thing he said when he was going over this. It probably has something to do with examining pedigrees of familial RB patients, noticing the inheritance pattern, and using some kind of tagging technology to figure out which gene was present in those with a greater tendency to get RB and absent in those without that tendency.
 * What are the properties of the protein product of the RB gene? List at least three biochemical properties
 * [Notice that the RB protein is a universal protein-- not just found in the retina.]
 * An inhibitor of the cell cycle that prevents proliferation.
 * Normally __hypophosphorylated__ (little PO4) to prevent proliferation.
 * __Hyperphosphorylated__ by __CDKs__ (cyclin-dependent kinases) to be turned __off__.
 * When turned __off__, allows normal cell proliferation.
 * When __kept__ off or inhibited, allows unchecked cell proliferation and carcinogenesis.
 * General note: RB protein works by binding to a variety of transcription factors.
 * Describe how the RB protein functions during the cell cycle and why it is important in cancer. You should be able to give an explanation of how the loss of RB may produce a malignancy.
 * As mentioned, functions to block G1 moving to S (replication) phase. Without RB, a cell has no 'brake' on its proliferation.
 * Notice that there are certain tissues that are particularly susceptible to the loss of RB-- that is, losing RB there has a particularly acute effect. Example is, obviously, the retina.
 * What is the hallmark of a tumor suppressor gene or anti-oncogene? You should be able to use the RB gene as an example
 * Prevents cells from proliferating by controlling cell cycle.
 * How were oncogenes discovered? You should be able to describe the method with at least three different examples
 * Discovered in oncogenic retroviruses (specifically Rous Sarcoma Virus in chickens). With one particular viral gene segment ( // v-onc // ), tumors are rapidly induced in the infected cells after infection; without it, integration into the host genome occurs without activation of oncogenes.
 * Examples: // v-src //, // v-erb // , // v-myc // , etc. Watch for the // v- // at the beginning of it. (as opposed to // c-myc // , which is an endogenous oncogene in the human genome.)
 * Often oncogenes mimic growth factor receptors to achieve their nefarious ends.
 * Method: take cells, put them in agar, watch for proliferation. Normal cells won't be able to proliferate (no anchorage to grow on)-- infected cells will proliferate regardless of anchorage (see characteristics of cancer cells, above).
 * What functions do the protein products of viral oncogenes perform? You should be able to give at least four examples of oncogenes of known function
 * RB protein is a target of some tumor viruses (eg. human papilloma virus), which produce proteins which inactivate RB (and/or p53) in the cell in which the virus has taken up residence.
 * This is a common theme: RB and/or p53 inactivation by viral proteins allowing rapid, unchecked cell proliferation.
 * Notice Karposi's sarcoma [HIV/AIDS] is also caused by RB/p53-inactivating proteins.
 * Notice also that __retro__viral-induced cancers (ie, resultant from viral reverse-transcription of their oncogenes into human DNA) are very rare in humans. Our viruses tend to just inactivate p53 and RB rather than encode oncogenes themselves.
 * Oncogenes:
 * As mentioned, the viral // src //, // erb // , // myc // , etc, sequences. Notice that viral copies of oncogenes tend to be more powerful effects than their endogenous counterparts.
 * // v-src // : phosphorylates various tyrosine residues in other proteins (similar to ABL in humans).
 * // v-erb // : mimics epidermal growth factor receptor (unregulated).
 * // v-sis // : mimics platelet-derived growth factor (unregulated).
 * Endogenous oncogenes are marked // c-onc // :
 * Notice that // c-onc // genes are part of normal functioning of human cells; therapy can't target all // c-onc //, just their overexpression.
 * // c-onc // genes need to undergo mutation before they become carcinogenic.
 * [Not on LOs but probably helpful:]
 * __APC__ gene product: keeps beta-catenin outside the nucleus; without APC, beta-catenin goes to nucleus and begins uncontrolled transcription of oncogenes (like // c-myc // ).
 * LOH in APC produces familial adenomatous polyposis (FAP), which leads to colon polyps and, eventually, metastatic colon cancer.
 * __BRCA1__ (breast-cancer gene 1): Its gene product forms the scaffold for protein assembly that "checks up on" the cell cycle to make sure that the DNA has replicated faithfully. When it's knocked out, the check on the cell cycle is removed.
 * Can either be familial (LOH as above) or sporadic.
 * Note that in sporadic cases, the mutation can be on other (unspecified) genes that regulate or have an effect on BRCA's expression.
 * __p53__ you may recall from Li-Fraumeni syndrome-- mutant or inactivated p53 is found in ~50% of all cancers.
 * Protein involved in DNA mutation repair at "checkpoint" in cell cycle.
 * Notice that mutations in p53 can be "dominant-negative;" that is, one bad copy of p53 can inactivate the other, good copy of p53.
 * Along with RB, one of the two major gene products knocked out by oncoviruses to produce cancers.
 * Why are oncogenes useful as molecular markers in prognosis? You should be able to give at least two examples of oncogenes that are currently being used and also include the evidence of why these are good markers.
 * The level of expression of oncogenes tends to correlate with the rapidity of the progress of the cancer.
 * One example: The level of expression of the // N-myc // gene is used in prognosis analysis for neuroblastoma.
 * Another example: Increased expression of the // HER2/neu // gene correlates with poor prognosis in breast cancer.
 * What is the difference between oncogenes and tumor suppressor genes? You should be able to describe the function of these two types of cancer genes and how mutations in them may combine to produce cancers.
 * Again: oncogenes promote cell proliferation, tumor suppressor genes inhibit it. If you have an over-activation mutation in an oncogene and an inhibiting mutation in a tumor suppressor gene, presumably you can have a real problem.
 * How can our knowledge of oncogenes and tumor suppressor genes be used for targeted therapy? You should be able to give an example of each.
 * Can design either small molecules or antibodies as therapy.
 * Targeting oncogenes:
 * Herceptin: drug antibody therapy against the HER2/erb2 oncogene product.
 * Small molecules: able to inhibit action of cancerous proteins by, among other pathways, binding to their active sites. Example is Gleevac, an ATP analogue, that inhibits ABL tyrosine kinase in patients with BRC-ABL translocation on the Philadelphia chromosome.
 * (BRC-ABL: translocation causing leukemia; resistant to radiation therapy. ABL is an ATP-dependent tyrosine protein kinase that PO4s various other proteins.)
 * Gleevac = specific to tyrosine kinase proteins: specific fit to their ATP-binding pocket.
 * Notice can use combined oncogene targeting therapy and radiation therapy: former makes the cancer more susceptible to the radiation.
 * Targeting tumor repressor genes:
 * You can inject RB directly into RB-negative tumors
 * You can use drugs that only kill cells with p53 deficiencies
 * You can use drugs which correct the mutant conformation of dominant-negative p53 proteins (see above).

=Composition of Cells=
 * Know typical values for the volumes of plasma (**3 liters** ), extracellular fluid ( **ECF, about 13 liters** + **another 5 for the 'third space'** ), and intracellular fluid ( **ICF, about 27 liters** ) compartments.
 * Of the roughly 45 liters of fluid in the body:
 * 99% is H2O
 * 0.8% is Na+, K+, Cl-
 * 0.2% is everything else.
 * Notice that there's roughly a 2:1 ratio between ICF and ECF.
 * 'Third space': fluid in GI tract, urine, CSF, sweat, etc. Effectively this is any fluid outside of cells but separated from interstitial fluid by epithelial cells. At the moment, more or less irrelevant.


 * Know the major difference in composition between ECF and ICF.
 * In ICF: mostly K+ cations (98% of K+ is inside cells) and a variety of anions (A-n, where -n is the average charge on the anions).
 * In ECF: mostly Na+ cations and Cl- anions.
 * [Types of ECF: interstitial fluid, lymph, plasma]
 * [Types of ICF: mitochondrial, nuclear, endoplasmic reticular, etc]
 * [For purposes of discussion, we're lumping all these together into ICF and ECF.]
 * Normal ion values:
 * __ICF__ (mM)
 * __ECF__ (mM)
 * __Membrane Permeability__ (yes/no)
 * Na+
 * 14
 * 140
 * (no) (yes, but actively pumped out)
 * K+
 * 145
 * 5
 * yes
 * Cl-
 * 5
 * 145
 * yes
 * A-n (anions)*
 * 126
 * ~0
 * no
 * H2O
 * ~55,000
 * ~55,000 (same as ICF)
 * yes
 * * = proteins, inorganic ions, etc.
 * Know the two most important functional properties of membranes, one conveyed by lipids, the other by channels and transporters.
 * Lipids are resistant to charge diffusion: that is, they can hold a differential charge on either side without having the charge leak across and balance out. There are two components to this:
 * One is that they are __impermeable to charged substances__ (ie water and ions).
 * The other is that they are 'strong' vs. electric force: they can withstand the large amount of electric force imposed by the charge differentiation. (note that if you increase the membrane differential by another 50 mV or so, this breaks down.)
 * Channels and transporters (proteins in lipid membrane) are the ways by which a cell gets around this impermeability. This allows a cell to be __selectively permeable__ and move ions back and forth in a controlled fashion.
 * // Channels // : allows passive diffusion out of or into cell. Notice:
 * They are generally __selective__ for a particular type of ion, and
 * They're usually '__gated__'-- that is, there's a particular type of stimulus that will cause it to open (particular receptor-binding ligands, stretch receptors, voltage changes, etc).
 * // Transporters // : actively pump ions out of or into cell.
 * Generally selectively bind large molecules for transport.
 * Can also pump ions against a gradient (ie. Na+ pump) using ATP.
 * Understand the routes by which a given substance can traverse a membrane.
 * See last two points (channels and transporters).
 * Notice, peripherally, that if the substance is lipid-soluble, it can just go straight into (and potentially through) the membrane itself (how it would have gotten to the membrane through the external aqueous environment is another question).
 * Identify physical forces that can determine the gating properties of ion channels.
 * Electric field (voltage-gated)
 * Mechanical (stretch, hair cells in cochlea)
 * Chemical (synaptic receptors)
 * Temperature (cutaneous temp receptors)

=Cell Volume Regulation=
 * Be able to determine which direction a given uncharged substance will move across a membrane, given its concentrations on the two sides.
 * Basic law of everything: substances tend to move from areas of high concentration to areas of low concentration. This probably is connected to the second law of thermodynamics (entropy, or randomness, or diffusion, is constantly increasing).
 * Notice that the diffusion of water down a gradient (ie. towards the area of low concentration) is called __osmosis__. This usually is discussed in the context of water flowing through a membrane that's impermeable to other substances, to balance out uneven osmotic concentrations of those concentrations on either side of the membrane.
 * Determine under a given set of conditions whether a cell will swell or shrink.
 * Notice that, given the relative concentrations of water vs. everything else inside of (and outside of) cells, __water__ alone is effectively going to determine changes in the volume of cells.
 * If the cell is immersed in water that's at a higher concentration (that is, it's more pure water and less solute) than the water inside the cell, the cell will __swell__.
 * If the cell is immersed in water that's at a lower concentration (that is, it's less pure water and more solute) than the water inside the cell, the cell will __shrink__.
 * Notice there's another way of saying this:
 * If the cell is immersed in water that has a __lower__ concentration of non-water stuff than the concentration of non-water stuff __inside__ the cell, the cell will __swell__ (water flows into cell to balance the concentrations).
 * If the cell is immersed in water that has a __higher__ concentration of non-water stuff than the concentration of non-water stuff __inside__ the cell, the cell will __shrink__ (water flows out of cell to balance the concentrations).
 * Notice that in pure water, our cells will burst (too much swelling!) because they're always going to have a higher concentration of solutes than their surroundings.
 * List the three mechanisms that different cells have evolved to keep from swelling and bursting.
 * (1) Make cell membranes impermeable to water.
 * This is not a popular option, since during cell division you're effectively going from 1 cell-full of water to 2 cell-fulls, and you've got to get water in there somehow to make up the extra volume. In other words: cells need to grow.
 * However, this can work sometimes in specialized systems (ie kidney cells when you need to retain water).
 * (2) "Brute force": build a wall around the cell which resists the osmotic pressure.
 * This is by far the most popular option-- all plant matter and pretty much everything other than animal cells.
 * The wall is permeable to water but resists the osmotic swelling force.
 * One problem is that it really limits mobility and morphology of the cell. The other problem is that it takes a lot of uptake and energy to maintain that wall.
 * (3) Balance the water concentrations osmotically.
 * Ie: put solute in extracellular fluid to make the water outside cells the same concentration as the water inside cells.
 * Notice that it doesn't really matter what solute you put in the ECF to dilute the water, as long as the solutes aren't able to go into the ICF (which would render the whole point moot).
 * Know which direction water will move across a semi-permeable membrane, given the solute compositions of the fluids on either side of the membrane.
 * Detailed answer: it depends on whether or not those solutes can cross the membrane and how fast they do so. See below, under "reflection coefficients," for details. Essentially if you have one solute that permeates the membrane more quickly than the other, the water will follow that solute first. If you have two permeable solutes, one on each side of the membrane, that both diffuse at the same rate, then the net water flow should be nada.
 * Simple answer: if the solutes can't cross the membrane, water will flow in the direction of greater solute concentration.
 * Know the difference between diffusion and osmosis.
 * Osmosis is the net inward movement of water across a semi-permeable membrane. Notice more detail in the definition on the first bullet point.
 * Diffusion is the more general movement of any solute from an area of high concentration of that solute to an area of low concentration of that solute.
 * Know which substances must move into or out of a cell in order for the cells volume to change.
 * Water alone is necessary and sufficient. To a first approximation.
 * Know the effect of having a membrane with different, non-zero permeabilities (ie, reflection coefficients less than 1) to different solutes.
 * ['reflection coefficient' = how permeable the cell membrane is to a substance: 0 = water, 1 = impermeable.]
 * Effectively, some things pass into cells more quickly than others, some things can't come into cells at all.
 * This has consequences: given the same concentrations of different __diffusable__ solutes on each side of the membrane, until they equilibrate out, one of them will likely diffuse __faster__ than the other one, which means that the balance of solute will __not__ be balanced throughout the diffusion process (which means the cell is going to either swell or shrink in the process as water follows the solute that diffuses faster).
 * Know the definitions of molarity, osmolarity, equivalents, and tonicity, and know how to convert between them.
 * Molarity: a standard measure of __concentration__ (grams of solute per liter of water).
 * Osmolarity: sum of molarity of all the solute particles. Effectively, you want to (ie. 1 M NaCl = 2 OsM since you have 1 M of Na+ and 1 M of Cl- when dissolved). Can calculate this for inside and outside the cell and compare them.
 * Notice that 1 M glucrose (C6H12O6) doesn't wind up being something crazy like 24 OsM, even though there's 24 atoms in each glucose molecule, how many atoms are in each, because sucrose doesn't dissolve into constituent atoms in water like ions in a salt do.
 * This can be used to determine whether a cell will swell or shrink, as noted above. Basically if you have a __higher__ osmolarity outside the cell than inside, the cell will __shrink__. If you have a __lower__ osmolarity outside the cell than inside, the cell will __swell__. If the osmolarity is the same inside and outside, the cell will stay the same size.
 * Important point: in a simplified situation (not comparing reflection coefficients or two opposing diffusing solutes), a solute that can cross the lipid membrane __does not change the volume of a cell at all__. It will go to equilibrium across that membrane, which means that the osmolarity both inside and outside the cell increases by the same amount, which means the __difference__ in osmolarity doesn't change at all-- thus no volume change.
 * But note caveat about relative permeabilities, above.
 * Tonicity: a measure of how a cell reacts to a given ECF solution. Also a way of describing the osmolarity of the __impermeable__ solutes in the ECF.
 * __Hypotonic__: any solution that makes a cell __swell__. (total __impermeable__ osmolarity = lower outside the cell)
 * __Hypertonic__: any solution that makes a cell __shrink__. (total __impermeable__ osmolarity = higher outside the cell).
 * __Isotonic__: any solution that doesn't make a cell shrink or swell- same __impermeable__ osmolarity as what's in the cell.
 * Equivalents: Take the ions that are the solute, break them down into osmolarity as above, then multiply the osmolarity by the charge on the ion. Basically gives you a feel for how much of a given osmolarity is acidic or basic. I find it unlikely he'll ask a question about this, but just in case.

=Membrane Potential I, II=
 * Compare qualitatively the relative strengths of electric and osmotic forces:
 * Electric forces are way crazy bigger than osmotic forces.
 * This is why a small number of ions can overcome a large concentration gradient.
 * Describe the two forces acting on an ion moving across a membrane:
 * Concentration gradient (higher concentrations diffuse to lower)
 * Electrical gradient (like charges repulse, opposite charges attract)
 * Define equilibrium potential:
 * For a given ion, the equilibrium potential is the potential across the membrane that that ion would 'like' to see exist in order to be in equilibrium.
 * If the membrane potential isn't at the equilibrium potential for the given ion, either the ion isn't able to permeate the membrane or there's some mechanism that keeps the ions from crossing the membrane effectively in order to equilibrate (ie, an ion pump).
 * Measured in millivolts.
 * Understand the difference between an equilibrium potential and a recorded membrane potential:
 * Membrane potential = electrical differential across the cell membrane. Empirical.
 * Equilibrium potential = calculated from Nernst equation for a particular ion. __Notice that the equilibrium potential of all permeating, non-pumped ions have to be equal to the membrane potential, at equilibrium.__
 * On membrane potential: by convention, notated about the inside of the cell with respect to the outside of the cell (so a negative membrane potential means there's a potential for positive ions to flow into the cell).
 * Know that each and every ion species has its own, independent equilibrium potential:
 * The equilibrium potential is dependent on particular electrical properties of the ion.
 * However, as above, note that for a non-pumped, permeating ion species (ie. Na+ or Cl-), the equilibrium potential is equal to the membrane potential for that membrane.
 * Understand that the number of excess anions in a typical cell is small compared to the total number of anions:
 * Doesn't need to be big to keep distinct ion concentration gradients in place.
 * Know that bulk solutions are always electrically neutral:
 * This, I think, is effectively because electrostatic repulsion doesn't allow charged solutions to stay together (see Betz's analogy about a one-liter box of cations needing a quarter of the weight of the earth to keep the lid on).
 * Technically this is not true across cell membranes - there's a small electric differential of ~-60 millivolts - but the electrical difference is small enough that we can get away with it.
 * What this means: in any given bulk fluid (ie. ECF/ICF) the electric charges must equal out.
 * This is able to keep concentration gradients away from equilibrium- that is, being electrically neutral takes precedence over being at a state of concentration equilibrium.
 * The cell is able to take advantage of this fact to maintain different compositions of ions inside and outside the cell.
 * Understand how an artificial cell can be in a state of equilibrium even though the concentrations of ions are not the same inside and out:
 * Okay. It's in a state of equilibrium because the electrostatic forces keep the ions from sliding along their concentration gradients.
 * Basically you've balanced the electrical equilibrium with the concentration equilibrium, This means that the cell isn't electrically neutral (though it's very close), and it also means that the cell isn't at a concentration neutrality (ie. different species of ions have different concentrations across the membrane).
 * Not to wax philosophical, but this is what allows us to be alive. Maintaining differences between things in the face of the 2nd law of thermodynamics - which drives things to a universal diffusion as the greatest expression of entropy - is what allows us to control fluxes in those differences, which in turn is the basis for pretty much all life as we know it, from arterial gas exchange to action potentials to which football team you root for.
 * Notice this electrical-vs.-concentration gradient thing does __not__ apply to uncharged solute atoms (electrostatic forces don't have any grip on uncharged atoms).
 * Be able to apply osmotic balance, charge neutrality, and the Nernst equation to calculate ion concentrations and the membrane potential of an artificial cell:
 * Nernst equation: Equilibrium potential is proportional to the natural log of the concentration of a given ion outside the cell over its concentration inside the cell.
 * ie: E is proportional to ln ([Iono]/[Ioni]) divided by the valence of the ion (+1 for Na+, -1 for Cl-, etc), where [Iono] is the concentration of the ion outside.
 * Approximation: E = 60/z * log ([Iono]/[Ioni]), where z is the valence (+1 for Na+, -1 for Cl-, etc).
 * (important) __Donnan equation__: [Ko][Clo] = [Ki][Cli]
 * Notes on possible test questions:
 * Dr. Betz seems to be fond of making charts where, given known and unknown concentrations of various ions inside and outside the cell, you fill in the blanks. There's a three-part process to solving these. This is what you need, not in any particular order:
 * (1) B__alance the electric charges //in each compartment//__ (ECF + ICF). ECF and ICF are always electrically neutral, more or less. So if inside a cell you've got 200 mosM of Na+ (valence +1) and 100 mosM of K+ (valence +1) and 50 mosM of proteins with a valence of -1, the total charge inside the cell right now is ((200*1) + (100*1) + (50*-1) = 250). To make it neutral, then, you need 250 mosM of Cl- (250*-1). If the valence of the proteins was -2, then the total charge inside the cell is ((200*1) + (150*1) + (50*-2) = 200). In which case you'd only need 200 mosM of Cl- to make it neutral. If this doesn't make sense, go find someone who understands it and threaten them until they verbally explain it.
 * (2) __Balance the osmolarity //between compartments//__ . If you add all the osmolarity of the ions inside the cell, it needs to be the same as the total osmolarity of the ions outside the cell. So if you've got 200 mosM of Na+, 100 mosM of K+, 50 mosM of proteins, and 250 mosM of Cl- in the ICF, total osmolarity = (200 + 100 + 50 + 250 = 600 mosM). This means that the total osmolarity in the ECF also has to be 600 mosM. So if you're missing one concentration in the ECF (in this example), just total the rest of them and subtract them from 600; what's less is what you have to have to balance the osmolarity. Again, if this doesn't make sense to you, find someone what can verbally explain it.
 * (3) __Use Donnan equation to get [K+] and [Cl-] for both compartments__. Because the product of [K+] and [Cl-] in the ICF is the same as the product of the concentrations of those ions outside the cell, if you know three of the concentrations, you can find the fourth. Ie: if AB = CD, and you know A, B, and C, D is pretty easy assuming you passed 9th grade algebra (which, for the record, I nearly didn't).
 * Something else he spent some time on in class: given a permeating ion's equilibrium potential (E) and the membrane potential (Vm) of the membrane, can you tell if there's an ion pump operating, and if so, in which direction it's operating?
 * This first part is easy. If a permeating ion's E isn't the same as the Vm, then there's a pump at work.
 * To figure out which direction it's going in, compare E and Vm.
 * If E is larger (more positive) than Vm, the ion wants to make the inside of the cell __more positive__.
 * This means if the ion's positively charged, it wants to go into the cell. Conversely, if the ion's negatively charged, it wants to go out of the cell.
 * If E is smaller (more negative) than Vm, the ion wants to make the inside of the cell __more negative__.
 * This means if the ion's positively charged, it wants to go out of the cell. Conversely, if the ion's negatively charged, it wants to go into the cell.
 * Try this on K and Na: K's E = -80, Na's E = +60. An average membrane potential is about -60.
 * Na's E is more positive than Vm, and it's a positively charged ion, so it wants to go into the cell.
 * K's E is more negative than Vm, and it's a positively charged ion, so it wants to go out of the cell.
 * Once you've figured out which direction the ion __wants__ to go, flip it. Then you know which direction the pump has to be pumping it to stop the leak in its preferred direction.
 * This is kind of a cheat sheet. But it's good to work through it on your own as well if you like mind pain.
 * Notice that, given the concentrations of ions inside and outside the cell, you can figure out E for those ions. So really, all you need to solve these problems is Vm and the concentrations of the ions you're looking at.

Other, random, notes: make membrane permeable to chloride but not sodium. Chloride atoms come into the cell, bringing a negative charge- when enough Cl ions are in there, electrostatic repulsion. Then when any Cl ion comes in along its concentration gradient, one gets kicked out by electrostatic repulsion. For K: same idea. Electrical gradient wants to keep K inside cell (since cell is negatively charged), even though concentration gradient wants to take K out of cell.

Na pump: transports Na out, K in.
 * In a real cell: the equilibrium potential for sodium is about +60. The equilibrium potential for potassium is about -80. The actual membrane potential is about -60. This means, since both K and Na are permeating, that there's a constant leak of K out of the cell (to make it more negative to approach equil potential) and a constant leak of Na into the cell (to make it more positive to approach equil potential). Thus rely on pump to pull back towards -60 as Vm. In other words, the current carried by sodium into the cell must equal the current carried by potassium out of the cell at equilibrium.

If you allow Na to come into cell (ie if you make the membrane more permeable to Na), get depolarization- cell membrane potential rises towards 60 mV.
 * Take-home here: any time you increase the membrane permeability of one ion, the membrane potential of the cell moves toward the equilibrium potential of that ion. And recall that the concentration gradient across the membrane determines the equilibrium potential for that ion. "The ion with the highest permeability wins."

Put another way: the ratio of the permeability of sodium to the permeability of potassium in a given cell is what determines the membrane potential of that cell. Notice that the permeability of a given membrane to an ion is largely determined by the number of open channels for that ion in that membrane. So if you have more open K channels than Na channels, your membrane is effectively more permeable to K than Na (and thus membrane potential will be closer to the equilibrium potential for K, -80, than it will be to the E of Na, 60).

=Acids, Bases, Buffers=
 * **Describe the law of mass action [le Chatelier's principle]:**
 * For any reversible reaction, the forward and reverse rates of the reaction are directly dependent on the concentrations of the __reactants__ and the __products__ // respectively //.
 * Ie: in A <--> B, if you increase the concentration of A, the rate of A -> B goes up. If you increase the concentration of B, the rate of B -> A goes up instead.
 * Equilibrium constant for a reaction Keq = [products] / [reactants].
 * Keq of water = 1.8 x 10-16 = [H+][OH-] / [H2O].
 * [H+][OH-] = 1 x 10-14.
 * This is universally, always, everywhere, true. No exceptions. Even for you.
 * In a neutral solution, [H+] = [OH-] = 1 x 10-7.


 * Define pH and pKa:

pH is the negative log of the proton concentration in a solution.
 * ie: pH = -log [H+].
 * In neutral solutions, [H+] = 1 x 10-7; log [H+] = -7, negative log = 7. **
 * That is: in neutral solutions, pH = 7, which is what you already know.

Side notes: histidine is the only amino acid side chain that protonates or deprotonates at physiological pHs- thus important for acid-base balance in body.
 * Notice this means that small differences in pH mean large differences in the concentration of protons-- which our bodies generally handle very poorly. **
 * Ka measures the tendency of a weak acid to dissociate in water.
 * pKa ** is the negative log of Ka for a given weak acid. **
 * The lower the pKa, the stronger the acid (more proton donation in water).
 * The higher the pKa, the weaker the acid (more proton acceptance in water).
 * **Write the Henderson-Hasselbalch (H-H) equation for any given weak acid or base:**
 * pH = pKa + log ([A-]/[HA])** . **
 * This means: pH = pKa + log ([proton acceptor]/[protein donor]).
 * Which means that, given the ratio of dissociated to undissociated acids in solution, you can calculate the pH of the system given the pKa, or can calculate pKa given the pH, or if you're given the pH and the pKa, can calculate the ratio of undissociated to dissociated acid in a given solution.
 * (ie: given two elements of that equation can get the third.)
 * Notice that when pH = pKa, the concentrations of deprotonated and protonated acid are equal.
 * (ie: when pH = pKa, the acid is half deprotonated and half protonated.)
 * **Define the H-H equation for the bicarbonate buffer system in extracellular fluid and use it to evaluate clinical lab data:**
 * pH = 6.1 + log ([HCO3-]/(0.3*PCO2))** . **
 * Essentially: the deprotonated bicarbonate (HCO3-) levels inversely vary with the partial pressure [levels] of CO2 in the blood.
 * What the hell that means: given the partial pressure of CO2 in the blood and the pH of the blood, can calculate the concentration of bicarbonate; or given the partial pressure of CO2 and the concentration of bicarbonate, can calculate the pH of the blood; or given the concentration of bicarbonate and the pH, can calculate the partial pressure of CO2 in the blood.
 * (ie: given two elements of that equation can get the third.)
 * **Important note: [HCO3-] needs to be measured in** millimolar** units; PCO2 needs to be measured in **millimeters mercury ** or **mm Hg** . **
 * Some background on all this mess: the buffering system (see below for details on buffering) in the body is predominantly made up of carbonic acid-to-bicarbonate: H2CO3 <--> H+ + HCO3-
 * This depends on the carbon dioxide levels in the blood-- dissolved CO2 reacts with water to form H2CO3.
 * This means that the equilibrium of the dissociation of H2CO3 is driven by the levels of CO2 in the blood (by le Chatelier's principle).
 * This works as a buffer, even though the nominal pKa of H2CO3 is 3.8 (thus would buffer optimally at 2.8-4.8, way outside physiological pH), because ion exchange in the kidney and input of CO2 changes the effective, actual pKa.
 * Define normal blood pH, [HCO3-] and pCO2 (please see your handout or learning objective):
 * Normal arterial blood pH: 7.34-7.44
 * Normal venous blood ph: 7.28-7.42 (lower due to higher [CO2])
 * Normal concentration of HCO3- in blood: 24 mM
 * Normal PCO2 in blood: 40 mm Hg
 * Normal concentration of CO2 in blood (he didn't ask, but it's in his practice problems): 1.2 mM
 * Describe how weak acids and bases work to buffer pH and define the pH range of maximal buffering capacity:
 * Effectively, you have a mix of weak bases and acids: the weak acids neutralize added bases without changing the pH much, the weak bases neutralize added acids without changing the pH much.
 * Optimal buffering capacity is determined by the pKa of the buffering acids/bases. There's a whole lot of titration chemistry behind this. Effectively there's a pH at which your buffer system can neutralize added acids or bases with very little change in overall pH, which is what you want to avoid a lot of big jumps in your blood pH (which would kill you): __this optimal buffering pH is plus or minus 1 pH unit from the pKa of the acid/base__.
 * Use the H-H equation to solve problems of how pH changes in defined buffers i.e. you should be able to determine how many equivalents of acid or base are needed to titrate the ionizable groups(s) of a weak acid or base from a starting pH to a final pH, given its concentration and pKa:
 * Quick way to think about this: every factor of ten that you have of deprotonated acid (A-) more than protonated acid (HA), you raise the pH by 1 over the pKa. If you have 1 HA molecule and 10 A- molecules, you just raised the pH by 1 from the pKa. If you have 1 HA and 100 A-, now the pH will be 2 plus the pKa.
 * Conversely, every factor of ten that you have of HA more than A-, you lower the pH by 1 from the pKa. So if you've got 10 HA molecules and 1 A- molecule, the pH of the solution is going to be the pKa minus 1.
 * So if you start out with a pH of 6 and the pKa is 6, to get to a pH of 7, you'll need to add in ten times the current amount of A- (which will add one to the pKa and result in a pH of 7). If you want to get to pH 5, you need to add in ten times the current amount of HA. pH 8: 100 times the current concentration of A-. pH 4: 100 times the current concentration of HA. Etc.
 * Again: find someone who gets this and beat or plead an explanation out of them if this is Greek to you.

Notice that the kidney controls a lot of pH by retaining or excreting HCO3- and NH4+.

Notice that the low pH in the stomach, which allows compounds to be easily protonated, allows drugs that need to be protonated to be absorbed into the cell to be so-

Remember: Higher levels of CO2 in the blood lower the blood's pH. This is because you make more carbonic acid, which drives the deprotonization of carbonic acid to form bicarbonate + a proton. These extra protons float around in the blood and lower the pH.

=Membrane Structure= has another order of magnitude of surface area over the intracellular membrane area. o Notice that under physiological pH, lipid bilayers spontaneously reform compartments to minimize contact of their hydrophobic tails with water.
 * Describe the lipid components of biological membranes and what differences exist between membranes of the different organelles
 * Lipid bilayers: roughly 5 nm thick, spanned by proteins.
 * Head groups: polar. Tail groups (inside the membrane): hydrophobic.
 * Classes of lipids in membranes:
 * Phosphoglycerides
 * Phosphotidylethanolamine
 * Phosphotidylcholine
 * Phosphotidylserine
 * Phosphotidylinositol
 * Sphingolipids
 * Cholesterol
 * All are synthesized at least in part in the ER and have polar and nonpolar ends.
 * __Phosphoglycerides__: have a glycerol-3-phosphate backbone with 2 fatty acyl chains (either saturated or mono- or polyunsaturated) at one end and a polar head group at the other end. What type of head group determines which phosphatidylglycerol it is (ethanolamine, inositol, etc).
 * __Sphingolipids__: have a 'sphingosine' (long acyl unit) with either of two polar head groups (which one it is distinguishes which type of sphingolipid it is) and an attached fatty acid chain.
 * __Sphingomyelin__: phosphocholine head group.
 * These are started to be made in the ER and finished in the Golgi apparatus.
 * __Cholesterol__: have a characteristic hydroxylated ring structure at one end, and a fatty acid chain on the other.
 * About 30% of all genomic proteins are membrane proteins
 * There are some double-membrane (mitochondria) and some single-membrane (nucleus) organelles.
 * Notice that there's roughly ten times the surface area of the plasma membrane contained as surface area of membranes within the cell; notice that the cytoskeleton
 * Bilayers are asymmetric. Notice that particular phospholipids don't flip from one side of the membrane to another without enzyme activity, but lots of lateral diffusion all the time.
 * [Not on LO's so learn at own risk:]
 * [Exoplasmic face: phosphotidylcholine, sphingomyelin, glycolipids (glycosylated lipids).
 * Cytoplasmic face: phosphoidylethanolamine, phosphatidylserine, phosphatidylinositol.]
 * Cholesterol is probably roughly equally distributed on both sides of the membrane.
 * Understand how concept of membrane fluidity and how membrane fluidity is modified
 * Membrane fluidity: the degree to which lateral motion is possible among adjacent phospholipids on a given side of the bilayer. Some lipids are anchored, some are free-floating within any given membrane.
 * Notice that different physical compartments of a membrane can have different degrees of fluidity.
 * Having acyl chains unsaturated makes the membrane more fluid (less tightly packed).
 * Cholesterol, when intercalated in membranes, stiffens the membrane and makes it less fluid.
 * Membrane composition determines thickness and curvature:
 * Membranes high in cholesterol also thicken the membrane by straightening out the nonpolar, hydrophobic groups inside the bilayer.
 * Curvature is dependent on the size of the polar head groups on both sides of the membranes.
 * Understand the many different ways proteins interact with membranes
 * [Cholesterol production: Proteins SCAP and SREBP, under high-cholesterol conditions, stay inactive in the ER. Under low-cholesterol conditions, SCAP and SREBP go on to the Golgi and wind up triggering nuclear receptors to synthesize more cholesterol.]
 * Different kinds of membrane proteins:
 * __Integral__ proteins (fully or partially embedded in membrane)-- can go multiple times back and forth through the membrane (multiple transmembrane domains).
 * Notice that transmembrane domains are usually alpha-helices, but can also have beta-sheet barrel formations (usually bacterial).
 * Can have transmembrane proteins that are only bound to one side of the bilayer.
 * Can have proteins that are only attached at their ends to the membranes.
 * __Peripheral__ membrane proteins: covalently interacting with proteins bound in the membrane, but not themselves embedded in the membrane.
 * Describe the importance of sphingolipids and cholesterol in biological membranes
 * Cholesterol: see above (thickness and nonfluidity).
 * Lipid rafts: formed primarily from sphingomyelin and cholesterol, thus extremely nonfluid-- effectively a lipid platform for various proteins. Used for signaling processes.
 * Although the different carriers and channels that allow molecules to be transported across membranes is discussed by Dr. Betz in his lectures, understand how these molecules' function should be integrated into the function of biological membranes.

=Membrane Fusion=
 * Understand the importance of sub-cellular protein targeting.
 * A cell has a lot of compartments through which it needs to move big proteins without compromising the membranes' integrity-- thus transport vesicles (membrane package-wrap, made from membrane of membrane-bound organelle of origin, containing cargo to be transported to target membrane-bound organelles).
 * This (vesicular transport) is the basic principle of transporting substances from one intracellular compartment to another.
 * Incorrect transport frequently results in disease: cystic fibrosis is due to a failure in chloride ion channel-protein transport.
 * Myelin/tubulin cytoskeleton elements: act as "highways" to transport vesicles throughout cell. "Motor" proteins use ATP to drive vesicles along these highways (actin for myelin, kinesin/dynein for tubulin). The particular motor proteins are specific for particular destinations-- one variety of kinesin may take a protein to one location, another to another, etc.
 * Know the basic principles of membrane and viral fusion:
 * Membranes don't just automatically merge when they get close together (which is good, otherwise things would not be well separated in the cell).
 * Two membranes: begin with them coated with water molecules. First you have to remove the water before you can fuse the lipids; then you need to overcome charge repulsion between lipids in order to allow a greater efficiency of merging. Also need to make sure these specific lipids should be merging in the first place (specificity of membrane fusion).
 * Early research: Botulin and tetanus toxins inhibit vesicle secretion by neurotransmitters. They do this by cleaving particular proteins: the SNARE proteins.
 * Know the function and structure of SNARE proteins:
 * Come in three shapes:
 * VAMPs: transmembrane domain at one end, with a helical domain in it.
 * Syntaxin: transmembrane domain, still a helical domain.
 * SNAP25: no transmembrane domain, two helical domains, fatty acid binding region that more or less acts as a membrane-binding domain.
 * (notice that there are different flavors of each protein, each of which can only bind to certain flavors of their counterparts.)
 * The helical domains of all three proteins serve to bind with each other-- form an aligned bundle or tetramer.
 * All __vesicles__ contain VAMPs. All __target membranes__ contain syntaxin and SNAP25.
 * When the vesicle nears the target membrane, the helical domains on the vesicle bind to those on the target membrane: these effectively press the two lipid layers together, squeeze out water and overcome charge repulsion, and promote the lipid fusion.
 * To do this, the helical binding needs to be extremely strong, and so it is.
 * This means a problem after the lipids have fused: how do you get apart the SNARE proteins once they've bound together so strongly?
 * Know regulation of SNARE-based fusion:
 * Enzyme that regulates the dissociation of SNARE proteins: //NSF// protein. Forms a kind of turning barrel around the SNARE complex and twists it apart with the help of a lot of ATP.
 * After being unwound, syntaxin becomes unfolded; need another protein, //Sec1//, to refold it properly.
 * __SNARE cycle__: VAMP on vesicle, syntaxin and SNAP25 on target membrane, helical binding and lipid fusion, NSF-mediated unwinding, refolding of syntaxin with Sec1.
 * Know the mechanism of viral fusion:
 * ["__Envelope viruses__" (influenza, ebola, HIV, etc): unlike other viruses, it has a membrane envelope around its capsid (capsid: protein structure containing genetic material).]
 * In order to infect a cell, the envelope needs to fuse with the target membrane.
 * Notice that the target isn't always the plasma membrane: influenza virus gets endocytosed first, then when it's inside the cell it goes after the endosome membrane.
 * Notice that when a cell becomes infected by an envelope virus, the cell makes viral fusogenic proteins that go to the cell's surface-- will put these fusogenic proteins on emergent viral capsids.
 * Mechanism is similar to SNARE-mediated fusion, but virus has only one protein:
 * Has a transmembrane domain as well as a non-transmembrane hydrophobic domain hidden inside the folded protein.
 * Binding to the cell surface causes the exposure of this hidden hydrophobic domain, which inserts into the host membrane.
 * The virus has a lot of these proteins on its envelope: effectively they bind the envelope to the host membrane.
 * The other feature of the protein is multiple helical domains: one they're bound to the host membrane, these domains bind strongly to each other and ratchet the envelope tightly onto the host membrane, squeezing out water and overcoming charge repulsion as per the SNARE mechanism.
 * Know the regulation of viral fusion:
 * Only thing I can think of that he mentioned was that influenza viruses are dependent on a cellular drop in pH to release their genetic material into the cell.

=Membrane Potential III=
 * Know the difference between equilibrium and steady state:
 * Equilibrium: Ion concentrations are not changing, inside or out, over time.
 * Steady-state: Ion concentrations are not changing, inside or out, over time because the cell is furiously working to keep them that way.
 * Equilibrium is Zen (no ions have any striving ideas to go in or come out).
 * Steady-state is a contained prison riot occurring at the same time as an attempted storming of the Bastille (lots of things want to go out, lots of things want to come in, the guards are beating everyone the hell up to keep them put).
 * In a cell at rest (steady state), given an ion's concentration inside and out, the membrane potential, and knowledge that the membrane is permeable to the ion in question, be able to determine whether or not a pump for that ion must exist, and if so, which direction it pumps the ion:
 * I went over this in detail at the end of the notes for "Membrane Potential II".
 * Understand that membrane potential depends on relative, not absolute, permeabilities to ion:
 * Example: a membrane is very permeable to sodium but not to potassium. The membrane potential then approaches the equilibrium potential of sodium (+60). If the membrane is very permeable to potassium but not to sodium, the Vm approaches the E of potassium (-80). If they permeabilities are equal, the Vm won't change - within a first approximation - no matter how much absolute quantity there is (note, however, that if the membrane gets really really permeable, the Vm will indeed change and approach 0 as the changing concentrations of the ions make their Es approach 0).
 * Understand that the short term determinant of membrane potential is not the Na/K pump, but relative membrane permeabilities:
 * Depending on the size and the number of channels in the membranes (larger cells with more channels are more vulnerable, quicker, to Na/K pump failure than others).
 * Define Driving Force of an ion:
 * The ion "wants" to have the membrane potential approach its own equilibrium potential. It will drive the membrane potential towards that equilibrium, aided or counteracted by other ions, which are also driving the membrane potential towards their own equilibrium potentials.
 * Understand why, in neurons and other excitable cells, membrane potential is sensitive to small changes in [K+]o, but not [Na+]o:
 * (Understand that [Na+o] is the sodium concentration outside the cell.)
 * (1) Because the membrane potential of the cell is nearly at the E of K+ anyway, the loss of extracellular Na+ (which brings the E of Na+ closer to 0) will make the Vm slightly hyperpolarized (closer to -80 mV) but have not much other effect.
 * (2) [K+]o is more sensitive because its starting concentration outside the cell are much smaller than Na+, and thus is much more sensitive to small changes in concentration.
 * Know the mechanisms by which calcium, glucose + insulin, Na/K ion exchange resins, and renal dialysis counteract the signs of hyperkalemia:
 * Hyperkalemia: Excess of potassium in blood (outside cells, in ECF). This changes the membrane potential of the cell because it alters the concentration gradient on which the electrical gradient of the membrane is based.
 * Effectively, this raises the equilibrium potential of K+, which in turn makes the membrane potential rise substantially (by ~20 mV for a 2% calcium efflux).
 * This is almost universally fatal. The reason it's so dangerous is that messing with membrane potentials is a good way to screw up action potential propagation, particularly in the pacemakers in the heart-- thus can't get heart contraction signals.
 * Caused by a variety of things: massively lysed cells, kidney failure, severe muscle damage.
 * Treatment:
 * Can stabilize cardiac conduction system by giving Ca2+
 * Can get cells to take up the K+ by giving insulin and glucose to stimulate Na-K pump. (Insulin gets glucose into the cell, where it activates the pump to work harder).
 * If you give alka-seltzer and alkylyze blood, can stimulate proton pumps to get K+ into cells.
 * Can use cation exchange resins: Effectively, Na+-lined "flypaper" for K+ in the blood, swapping out Na+ for K+.
 * Can use renal dialysis to pherese excess K+ out of blood.

=Membrane Transport=
 * [Transporters: Proteins in the membrane that move specific substances, as in moving particular large molecule or pumping ions against their gradients.
 * Know the difference between primary and secondary active transport:
 * Primary active transporters: use ATP for energy source.
 * Ie: Na+-K+ pump, Ca2+ pump, proton pumps.
 * Secondary active transporters: don't use ATP. Uses energy derived indirectly from primary pumps.
 * Amino acid transporters: intake AA into cells; derives energy from leakage of Na+ into cell (slight amount of energy released by Na+ leak).
 * H+-to-Na+ exchange (protons out powered by energy of Na coming in).
 * Define cotransport and exchange transport:
 * Cotransport: secondary active transporters that move different solute species in the same direction.
 * Exchange transport: secondary active transporters that move solutes in opposite directions.
 * Na-Ca pump: Na+ in cell, Ca2+ out of cell (or, sometimes, the other direction)
 * A note about calcium: [Ca2+] inside the cell is kept extremely low (about 0.1 micromolar; about 10,000 times lower than outside the cell).
 * Thus: calcium has very high equilibrium potential (~120 mV). This allows single-channel openings to allow Ca2+ in can have large effects inside the cell.
 * The Na-Ca exchanger effectively works by balancing the favorable energy from 3Na+ entering the cell with the unfavorable energy from Ca2+ exiting the cell.
 * Notice that this can change direction depending on the current state of the membrane potential and the concentrations of Ca and K in the cell.
 * Be able to determine which direction a non-electrogenic secondary active transporter will operate, given the concentrations of the participating ions inside and out:
 * Electrogenic: one cycle produces a net charge transfer across the membrane. Thus non-electrogenic are transporters that don't change the net charge across the membrane. In this case, my guess would be that they tend to follow the direction of ion concentration (see the theoretical H/K exchanger below).
 * Know that electrogenic secondary active transporters can reverse direction, depending on membrane potential.
 * (see under Na-Ca pump above)
 * Understand the non-existence, but conceptually useful idea, of the H/K exchanger:
 * If you drop the proton concentration in the blood, can promote cell uptake of K in exchange for protons going out into the blood, and vice versa. The H-K exchanger doesn't actually exist as an entity, but you can think about it like that.
 * Essentially: if you have excess K, you can give bicarbonate (HCO3-) to decrease the total H+ concentration in the blood. When that happens, the exchanger that switches K for H swaps out K in the blood for H in the cell, thus making the proton concentration in the cell go down and the potassium concentration in the cell go up. This effectively takes K out of the blood, treating hyperkalemia.
 * Know how cells can concentrate glucose inside, even though glucose transporter cannot pump glucose against its concentration gradient
 * Inside cell, glucose gets phosphorylated, which makes it unable to fit into the glucose transporter-- thus glucose flow is more or less unidirectional into the cell. Notice that glucose transporters are normally sequestered within vesicles in the cell until insulin signals the cell to make those vesicles merge with the cell surface and transport glucose into the cell.
 * Notice that the CHF drug Digitalis blocks the Na-K pump. It does this in order to increase the contractile strength of the heart muscle; what it does is raise the effective concentration of Na+ in the cell-- which then renders the Na-Ca pump less effective, allowing more Ca2+ to stay in the cell, increasing the strength of the muscle cell contraction.

=Epithelial Transport=
 * Epithelial cells: cells which sequester 'third space' fluids from extracellular fluid.
 * These cells are __polarized__: not electrically, but meaning that they transport in only one direction (apical to basolateral).
 * Side of epithelial cells facing the third-space fluid is called the '__apical__' or mucosal membrane; the side facing the ECF is the __basolateral__ or serosal membrane.
 * The basolateral membrane is generic across the entire surface.
 * The apical membrane is specialized and compartmentalized (with tight junctions) for special functions.
 * Mainly, these involve transporters of various kinds.
 * Tight junctions: here, protein-based 'glue' that holds nearby cells together but blocks lipid exchange with the basolateral membrane.
 * Understand the generic transport mechanisms for NaCl and water into the blood:
 * NaCl: Na+ picked up by the apical membrane (high permeability of apical membrane for Na+) and is transported into the cell, then is pumped out by the Na/K pump out the basolateral membrane. Cl- passively follows the Na+ to equalize the electrical potential.
 * Water follows the influx of NaCl into cells to balance osmolarity.
 * Notice all this is passive (no ATP usage) absorption.
 * Know the basic transport mechanisms by which glucose and amino acids are absorbed into the blood:
 * Epithelium in GI tract: AA, sugars, and glucose are pumped (secondary active transport) through the apical membrane and diffuse out the basolateral side passively into the blood.
 * AA and sugars are picked up by their target cells by secondary active transport mechanisms.
 * Glucose, as mentioned before, diffuses into cell along its gradient (but is phosphorylated in the cell to prevent efflux).
 * Know the major differences between 'tight' and 'leaky' epithelia:
 * Tight: more junction proteins, tighter seal between cells. Used particularly in the distal tubules of the kidney.
 * Loose: less junction proteins, looser seal between cells.
 * Tight: "fine tuning"-- strictly controlled substance transport at lower levels, don't want backflux.
 * Loose: quantity over quality (needs lots of transporters, not too picky about equal amounts).
 * Know that four important substances - water, O2, CO2, and urea are never pumped, but always move passively down their concentration gradients:
 * You can open and close aquaporin channels to allow the water to come in or go out, but it always flows along its gradient.
 * Understand the relative roles of the G.I. tract (minimal) and kidney (extensive) in excreting non-volatile metabolic wastes and regulating ECF composition:
 * GI tract excretes (in feces) largely things you couldn't absorb in the first place. GI tract absorbs pretty much everything it can that comes in to it indiscriminately (thus its small role in excreting waste).
 * However, GI tract does excrete dead red blood cells (excreted into the GI tract from the liver), which is significant.
 * Kidney concentrates waste very well in the urine- gets rid of approximately 0.5 moles of non-volatile (nongaseous) metabolic waste a day, mainly end-products of nitrogen metabolism (urea) and protons.
 * Given two of the following, be able to calculate the third: apical membrane potential, basolateral membrane potential, trans-epithelial potential:
 * Trans-epithelial membrane potential = basolateral membrane potential - apical membrane potential.
 * Notice trans-epithelial is abbreviated PD.
 * One thing to remember here is that the membrane potentials of the apical and the basolateral membrane will be different (different ion permeabilities, etc).
 * The other thing to remember is that membrane potentials are always written in the form of describing the inside of the membrane with respect to the outside-- ie, in a membrane potential is -10 mV, the inside of the cell is 10 mV more negative than the outside.
 * Know the basic process by which some epithelial cells secrete (rather than absorb) fluid.
 * There's a chloride-selective channel in the apical membrane in some epithelial cells, cAMP-gated on the inside of the cell to open and allow the efflux of Cl- back out of the apical membrane (which takes Na+ and water with it).
 * This process is driven by receptors on the basolateral side of the epithelial membrane that are triggered by acetylcholine to open those gates.
 * The reason the Cl- channels excrete Cl- instead of intaking it is that there's an non-electrogenic pump on the basolateral side that uses Na+ leakage to import Cl- into the cell.
 * Effectively you're putting watery/serous substance out into the epithelial secretions (mucus, etc).
 * This is the basis for cystic fibrosis: the chloride channel isn't properly implanted in the cell membrane, and thus no dilution of mucus secretions are possible.
 * This is also the basis for cholera: the cholera toxin gets into the cell and opens the Cl- channel without regulation- which leads to a massive efflux of water out the apical membrane into the epithelial system (diarrhea, etc).
 * Understand the main routes of excretion of metabolic wastes - CO2 and urea, in particular.
 * CO2, unsurprisingly, is excreted in the alveoli of the lungs.
 * Urea is excreted in the urine after being picked up out of the blood in the kidneys.

=Action Potential I=
 * Understand how the passive electrical properties of axons render them poor conductors of electrical signals over distances greater than a few millimeters:
 * The electrical resistance in cytoplasm is high- also the electrical insulation provided by the cell membrane is poor. Unassisted passive electrical conductance would allow the signal to completely decay before it reached its target.
 * Describe the analogy between an electrical 'booster station' and an action potential:
 * Essentially, at various "stations" along the axon, a power source (the gradient of Na+, that is, potential chemical energy) 'boosts' the electrical signal back its original strength, based on the 'readings' of the signal strength by voltage-sensing receptors.
 * In axons this 'reading' is done by __voltage-gated sodium channels__.
 * Know how changes in membrane resistance, membrane capacitance, and internal (axial) resistance affect the passive spread of voltage along an axon:
 * Okay. The cell membrane acts as a capacitor between the ICF and the ECF. Notice that as soon as current reaches the ECF it is effectively lost-- it's no good for signal transmission.
 * The ion channels in the cell act as resistors that slow the flow of current from the ICF to the ECF.
 * The resistance of the cytoplasm in the axon itself acts as a resistor that slow the flow of current along the ICF (in the direction you want the impulse to go, namely down the axon).
 * What this means if you want an axon to propagate quickly:
 * (a) You want the capacitance to be low (high capacitance delays the flow of current down the axon, since it's the first place your current goes).
 * (b) You want there to be lots of resistance to current leaving the cell (thus closing the ion channels).
 * (c) You want as little resistance as possible to current traveling down the axon.
 * The way the cell does this is (a) wrap the axons in myelin, which effectively increases the thickness of the membrane and thus drops the capacitance, speeding up the electrical propagation; (b) close ion channels to pump up resistance to current leaving the cell; and (c) increase the diameter of the axon, which increases current flow and effectively lowers internal (along-the-axon) resistance.
 * If this doesn't make much sense to you, you're in good company, or at least in mine. Just remember: thick membranes = low membrane capacitance = faster AP transmission. Fewer on channels = high membrane resistance = faster AP transmission. Larger-diameter axon = lower internal resistance = faster AP transmission.

=Action Potential II, III=
 * [Features of APs:]
 * (1) Propagated
 * (2) Vm reverses
 * (3) Brief (<1 ms)
 * (4) Threshold
 * (5) Refractory
 * (6) Accommodation
 * Describe the positions of the activation and inactivation gates in sodium channels during an action potential:
 * Sodium activation gates (m gates** ) are 'normally' closed at rest; they open (thus making the membrane more permeable to sodium) in response to a small depolarization (ie inside of the cell gets more positive).
 * Notice that the // m // gates are on the // outside // of the cell.


 * Sodium inactivation gates (**h gates** ) are 'normally' open at rest; they close (thus making the membrane less permeable to sodium) in response to the large depolarization that occurs after the //m// gates are opened. Notice there's a lag time between the point at which the //m// gates open and the point at which the //h// gates close (which allows the flood of Na+ to fully depolarize the cell to generate the AP). **


 * Notice that the //h// gates are on the //inside// of the cell.
 * Notice that there are also voltage-gated K+ channels that allow the cell to repolarize much faster after firing an AP (allow K+ out of cell to restore Vm after influx of Na+). These open around the time the AP signal is at the peak of its intensity (ie. when the cell is approaching maximum depolarization).


 * **Understand that intracellular concentrations of sodium and potassium do not change much after a single action potential:**

The flow in of Na+, and the flow out of K+, don't put much of a dent in the overall stores of both of those ions in the cell. Therefore an axon can fire multiple times before it needs to equilibrate its Na/K levels with the Na-K pump.


 * [Length constant λ = length along axon it takes for amplitude of electrical signal to decay to 37% of its original strength. In cellular axons this is about 1 mm.]
 * [Time constant = time it takes for an axon's signal to reach its maximum strength.]
 * Understand the role of the sodium/potassium pump during the action potential:
 * During the AP, the Na-K pump does pretty much nothing important. It's needed down the road to make sure that the Na-K concentrations are back to their proper levels (see accommodation below), but in the short term it's more or less irrelevant.
 * Describe the mechanisms underlying the refractory period of the action potential:
 * Recall that there are //two// refractory phases: the __absolute__, during which the cell will not fire an AP no matter what the stimulus, and the __relative__, during which the cell needs a much larger stimulus than normal to fire another AP.
 * This is due to the fact that the //h// gates (Na inactivation gates) require some time before they can reopen and allow Na into the cell (thus even if //m// gates open, the Na+ can't get into the cell).
 * During the absolute refractory phase, the //h// gates are more or less all shut.
 * During the relative refractory phase, the //h// gates are not all shut, but enough of them are that it will require more depolarizing stimulus to trigger the Na+ positive feedback cycle.
 * Notice that the K+ channels also contribute to the refractory period: it takes them a little while to close after repolarizing the cell, which help make the cell briefly hyperpolarized during the refractory period and makes it harder for the cell to become depolarized enough to trigger another AP.
 * Notice that the refractory period is the basis for the unidirectionality of an AP: as the impulse travels down an axon, it can't travel backwards because the portion of the axon just behind it (the one that just fired the AP) is in its refractory period.
 * (note: here I use "unidirectionality" to refer to the fact that an AP can't go backwards. Remember, thought, that if you start an AP in the middle of an axon, it goes in __both__ directions down the axons. One way of thinking about this is that __the direction of conduction down an axon__ isn't unidirectional, but __the direction a given AP travels__ is.)
 * **Describe the mechanisms underlying accommodation of the action potential:**
 * **Accommodation** : If a progressively larger current is applied to the cell slowly, often an AP won't fire-- if the depolarization is slow, the inactivation ( // h // ) gates have enough time to close before the AP has a chance to get going.
 * This is the basis for the problem with hyperkalemia: you wind up with a slow depolarization of the membrane that prevents APs from firing by slowly activating the // h // gates and preventing sudden depolarization by positive feedback.


 * This means that the more APs a cell fires, the more steadily depolarized it becomes. This, in turn, means that after a certain point of depolarization, the //h// gates (inactivation) are shut, and stay shut, meaning that the cell can't fire more APs until the Na-K pump restores its normal concentrations.

When this happens depends on the axon's diameter. Basically, the diameter of the axon determines its surface-area-to-volume ratio: the larger the axon, the smaller this ratio, which means that its Na-K balance is disturbed relatively less per AP than a smaller-diameter axon.
 * Thus: large-diameter axons accommodate more slowly; small-diameter axons accommodate more slowly.]


 * Define threshold for an action potential:
 * AP **threshold** is the point at which the incoming current of Na+ __equals__ the outgoing current of K+ (as Na+ comes in, K+ wants to come out more strongly). If the incoming current (Na+) gets just a little higher, the cell will enter the positive-feedback loop of depolarization.
 * A more intuitive way of thinking about it is that the threshold is the point at which just a hair more depolarization will probably make the cell fire an AP and just a hair less depolarization will probably make the cell return to normal polarization levels.
 * Understand the explosive, positive-feedback nature of the rising phase of the action potential:
 * If a cell is depolarized past its threshold point, the depolarization of the cell causes Na+ channels to open (//__m__// __gates__), which causes the cell to depolarize even faster, which causes more //m// gates to open, etc, etc.
 * Describe how action potential propagation relies on voltage-gated sodium channels acting like molecular 'booster stations'
 * See "Action Potential I." Effectively the passive propagation of the AP is sufficient to depolarize the next segment of axon enough to cause that second segment's //m// gates to open, causing another AP-- and so on down the axon.
 * [Basis for anesthestic: block //m// gates so that depolarization can't happen.]
 * [Notice: __Difference between small and large nerve fibers__:]
 * Small (ie pain and temperature fibers):
 * Slower AP conduction.
 * Have a high threshold to extracellular stimulation (harder to trigger AP).
 * Lower safety factor: easier to block //m// gates with anesthetic.
 * Large (ie touch receptor and positional fibers):
 * Faster AP conduction.
 * Have a lower threshold to extracellular stimulation (easier to trigger AP).
 * Higher safety factor: harder to block //m// gates with anesthetic.
 * Know why action potential propagation is much slower than the velocity of light:
 * Aside from the fact that it relies on the movement of ions (not massless particles), the fact that it's moving through a fluid with resistance as opposed to through a vacuum or through low-resistance copper wire, and the fact that it relies on a slow threshold-gated signal for propagation? No idea.
 * Know what myelination does to axonal membrane resistance and membrane capacitance, and why these changes increase conduction velocity
 * __Myelination__ effectively increases the __capacitance__ of the axon (making a signal go __faster__, no waiting for the capacitance to build) and increases the membrane __resistance__ (making a signal go __farther__, no leaking of ions out of the membrane).
 * Describe refractoriness, and explain how it prevents an action potential from reversing its direction of propagation:
 * See above, under "refractory period."
 * Understand why demyelination can slow or block action potential conduction:
 * Well, if myelination allows the AP to go faster through increased capacitance, demyelination can make an AP go slower. Likewise, if myelination allows an AP to go farther through increased membrane resistance, demyelinated can make it go a smaller effective distance, which may not be far enough to reach the next node of Ranvier and set off the next AP.
 * Describe the effect of extracellular calcium ions on action potential threshold:
 * Ca2+ in the ECF normally remains bound to negative charges on the outer surface of the cell.
 * However, in hypocalcemia, [Ca2+] in the ECF goes down, which allows some of those negative charges to go unbound. This makes the outside of the membrane, at particular places where the Ca2+ is thin, selectively more negative.
 * As the outside of the membrane gets more negative in places, the potential difference across the membrane at those places goes lessens (ie, it depolarizes), thus triggering the //m// gates to create an AP without normal signaling.
 * Note the difference between hyperkalemia and hypocalcemia:
 * Hyperkalemia: excess of [K+] in ECF causes gradual depolarization of the entire membrane, which allows //h// gates to shut and stay shut, preventing APs from being generated.
 * Hypocalcemia: insufficiency of [Ca2+] in ECF caused localized depolarization of the //outside// of the membrane, which causes //m// gates (which are on the outside) to open and cause APs to be generated erratically.
 * [First sign of this is often "Trousseau's sign": contraction of the flexors in the elbows and wrists.]
 * Know the effect of axon diameter on conduction velocity, threshold to extracellular stimulation, safety factor for conduction, and likelihood of being myelinated
 * //Axons with a larger diameter conduct impulses more quickly.//
 * Interesting aside: notice that, given unlimited space, we'd want all of our axons to be huge-diameter to get really quick information to our brains. But given limited space, there's a tradeoff between //size// of axon (faster signal) and //number// of axons (more information in a slower signal). So some of our axons are really big (the get-out-of-the-way-of-the-bus axons) and some are smaller but more numerous (the ponder-the-meaning-of-life axons).
 * __Safety factor__: the fact that we have a lot more Na+ //m// gates than we really need to get to threshold and generate an AP of sufficient size in a given axon.
 * Means faster conduction is possible (depends on # //m// gates).
 * Also means can send APs down multiple branches of an axon.
 * Threshold to extracellular stimulation: As mentioned, large-diameter axons have a lower threshold to stimulation, while small-diameter axons have a higher threshold.
 * Smallest axons are unmyelinated: actually it winds up being more efficient this way.
 * Know the mechanisms by which calcium, glucose + insulin, Na/K ion exchange resins, and renal dialysis counteract the signs of hyperkalemia:
 * See "Membrane Potential III."