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Null hypothesis (H0) - the hypothesis to be tested, typically a statement of "no difference/effect/association"; this is the hypothesis that is assumed true unless the test produces sufficient evidence to the contrary. Alternative hypothesis (H1 or Ha) - the hypothesis that contradicts the null hypothesis. The alternative hypothesis can be two-sided or one-sided; this should be decided before beginning analysis of the data. - Two-sided (≠): Used when study is interested in any deviation from the null. More commonly used than one-sided. - One-sided (<, >): Used if prior evidence or the nature of the study directs the focus towards a deviation in just one direction. A one-sided test increases the power to detect a significant effect in one direction, but does not account for deviations in the other direction at all. Thus, deciding to use a one-sided test requires careful consideration. OK: A newly developed drug is considerably cheaper than the option currently on the market. The researcher may choose to utilize a one-tailed test that will strongly detect if the new drug is LESS effective. The other tail need not be addressed because being more effective or merely equally effective are both fine, as the main positive point of the new drug in this case is its increased affordability. Not OK: A researcher wishes to demonstrate that a new drug is more effective than the current one, and chooses a one-tailed test in order to maximize detection of improvement in the data. However, this approach would fail to account for the possibility that the new drug is less effective. 2. Define and interpret p-values. What the p-value is: The probability of observing a result/difference as extreme as, or more extreme than, those observed in the studied sample(s), assuming that the null hypothesis is true. What the p-value is NOT: The probability that the null hypothesis is true. (See #6 in the practice problem set for this lecture.) How do you obtain the p-value? After calculating the test statistic (t or chi-squared), refer to a table or computer program that provides the corresponding p-value. Graphically speaking, the p-value is the area under the t or chi-squared distribution curve (probability density function) that lies beyond the calculated test statistic.

(For more on interpretation of p-values, see objective #4 regarding statistical significance.) 3. Recognize appropriate hypothesis tests for means. (i.e., continuous variables) Paired samples: Matched pairs (based on age, ethnicity, sex, etc.) or individual subjects “paired” with themselves; use paired t-test Two independent samples: Ex., subjects randomly assigned to one of two treatment groups; use 2-sample independent t-test More than two groups: Use ANOVA (ANalysis Of VAriance) models 4. Interpret statements of statistical significance. General guidelines (for typical significance level alpha = .05):

p <= .001 very highly significant .001 < p <= .01 highly significant .01 < p <= .05 significant p > .05 non-significant .05 < p <= .10 borderline significance Consider the typical wording of a conclusion statement in a research article: "The data demonstrate that there is a statistically significant difference between X and Y (p-value provided)." Avoid absolute statements (see practice problems #7 and #8) and always keep the definition of p-value in mind. 5. Distinguish between the statistical significance of a result and its importance in clinical application. Statistical significance: The test results are unlikely to occur by pure chance. Clinical significance: The test results indicate a difference/improvement/disparity that is meaningful from a healthcare standpoint. A result can be statistically significant and clinically insignificant (ex., a drug that is statistically significant in its performance vs. a placebo, but doesn't make enough of a difference in terms of treatment to be of any use in the clinical setting), or it can be statistically insignificant but still have potential for clinical significance (ex., statistical insignificance was due to insufficient sample sizes; requires further investigation). 6. Recognize when proportions are appropriate summary statistics. Proportions are useful when dealing with experiments with two outcomes (success vs. failure, yes vs. no, etc.). Sample proportion can be used to estimate population proportion (probability) of a given outcome. 7. Compute and interpret proportions and associated confidence intervals. (For equations used for calculation of the above, see handout or lecture slides.) Interpretation of sample estimate of population proportion: We estimate that X% of [the population in question] will have [outcome of interest]. –or– The estimated probability of [outcome of interest] is X%. Interpretation of 95% confidence interval (z = 1.96) for proportions: We are 95% confident that the true proportion of [outcome of interest] in [the population in question] is between [lower limit of confidence interval] and [upper limit of confidence interval].

8. Understand the two-by-two table format for displaying categorical data. Variable B Variable A Yes No Totals Yes a c a+c No b d b+d Totals a+b c+d n

Can test for association between variable A and variable B using 2 x 2 chi-squared test that compares observed values to the expected values assuming the variables are independent (i.e., assuming the null hypothesis of no association). 9. Distinguish between when to use a t-test and when to use a chi-squared test. t-test: Use to compare continuous outcome variable between groups (involves means) chi-squared test: Use to compare categorical outcome variable between groups (involves proportions) 10. Interpret p-values and statements of statistical significance for chi-squared tests. Follow same guidelines of interpretation as for t-tests. (See objectives #2 and #4.)

Microanatomy: Protein Synthesis and Secretion (Or, free vs bound ribosomes and Golgi complex details) 8/22/11 1) Describe the general mechanism of protein synthesis, and where it takes place in epithelial cells. Protein synthesis takes place on ribosomes. The ribosome life cycle a) Ribosomal RNA synthesized in nucleolus b) Ribosomal proteins synthesized in cytoplasm c) Ribosomal proteins imported to nucleolus, two subunits assembled (The two eukaryotic ribosomal subunits are 40s and 60s, useful to remember. Also useful numbers for the CF sweat test: <40mosm is negative, >60mosm is positive.) d) Ribosomal subunits are transported to cytoplasm. e) Small subunits identify and bind to mRNA f) Large subunits join to complex to form the ribosome. (The two eukaryotic ribosomal subunits assemble to form an 80s protein. Minimum sweat needed for the sweat test is 75 mg.)

FROM 2011 FIRST AID FOR THE USMLE STEP 1 Protein Synthesis Initiation Activated by GTP hydrolysis, initiation factors help assemble the 40s ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal subunit assemble with the complex. Elongation 1. Aminoacyl-tRNA binds to A site (except for initiator methionine) 2. Ribosomal rRNA (ribozyme) catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site. 3. Ribosome advances 3 nucleotides toward 3’ end of RNA, moving peptidyl RNA to P site (translocation) Termination Stop codon is revognized by release factor, and completed protein is released from ribosome. Mnemonics Eukaryotes: 40S+60S=80S (Even) prOkaryotes: 30S+50S=70S (Odd) ATP = tRNA Activation GTP = tRNA Gripping and Going places (translocation) Clinical Relevance Many antibiotics act as protein synthesis inhibitors Aminoglycosides inhibit formation of the initiation complex and cause misreading of mRNA Chloramphenicol inhibits 50S peptidyltransferase Macrolides and clindamycin bind 50S, blocking translocation.

Ribosome location Free ribosomes in cytoplasm vs. Bound ribosomes on ER. Both form polyribosomes (polysomes).

2) Diagram and list the basic functions of the RER, SER, and Golgi complex. Rough Endoplasmic Reticulum

FROM 2011 FIRST AID FOR THE USMLE STEP 1 Nissl Bodies (RER in neurons) synthesize enzymes (e.g. ChAT) and peptide neurotransmitters. Mucus-secreting goblet cells of small intestine and antibody-secreting plasma cells are rich in RER.

Smooth Endoplasmic Reticulum Irregular network of membrane-bound tubules. Functions in steroid hormone synthesis, drug detoxification, and release and recapture of calcium ions for muscle contraction.

FROM 2011 FIRST AID FOR THE USMLE STEP 1 Liver hepatocytes and syeroid hormone-producing cells of the adrenal cortex are rich in SER.

Golgi Complex

FROM 2011 FIRST AID FOR THE USMLE STEP 1 Golgi apparatus 1. Distribution center of proteins and lipids from ER to plasma membrane, lysosomes, and secretory vesicles. 2. Modifies N-oligosaccharides on asparagine. 3. Adds O-oligosaccharides to serine and threonine residues 4. Addition of mannose-6-phosphate to specific lysosomal proteins targets the protein to the lysosome 5. Proteoglycan assembly from core proteins 6. Sufation of sugars in proteoglycans and of selected tyrosine on proteins. Vesicular trafficking proteins: COPI: Retrograde, Golgi --> ER COPII: anterograde, RER Golgi Clathrin: trans-Golgi lysosomes, plasma membrane endosomes

3) Distinguish between regulated and constitutive secretory pathways. Constitutive Secretion (default pathway): Usually continuous, needs no special stimulus. Can be upregulated or downregulated. Regulated Secretion: Requires an extracellular stimulus. Until then, proteins to be secreted are

concentrated into secretory granules.

**Genetics: Human Inheritance I
**

Bold indicates a learning objective term/definition.

Methods of Inheritance: Locus – genetic location on a chromosome Allele – each individual has two alleles at a locus, one copy from each parent Genotype – the record of an individual’s alleles, genetic information. Often written AA (homozygous dominant), Aa (heterozygous, sometimes called carrier), or aa (homozygous recessive) for a given trait. Autosomal – No effect of gender, males and females equally likely to be affected and to pass on trait. Sex-linked – Affected by gender, gene resides on one of the sex chromosomes. Dominant – One copy of gene confers trait. Affected seen in all generations, does not skip. About half of offspring of one affected and one non-affected parent will have the trait. Also called “true dominant.” Co-dominant – In a heterozygote, both alleles are expressed as individuals. (ex: blood group AB) Semi-dominant – Subjects who are homozygous dominant more severely affected than heterozygotes. Recessive – Two copies of gene needed to confer trait. Often appears to skip generations, or a single generation may be affected. If both parents carriers, ¼ of offspring affected. X-linked – sex-linked trait on the X chromosome. More often expressed in men (only one X), will never see male-to-male transmission. Women are usually carriers (Xx) but could also show disease if trait is dominant, they are homozygous recessive, or there is skewed Xinactivation. Y-linked – sex-linked trait on the Y chromosome, very rare. Only male-to-male transmission, no skipped generations. Mitochondrial inheritance – all inheritance is passed from mother to child, regardless off child’s gender. No transmission from father to any offspring. Random (disassortative) mating – mate selection is not based on similarities or biased by background, physical ability, etc. Founder effect – High frequency of a previously rare allele because population was started by small number of founders. Also called a “bottleneck.” Mosaicism – Individual has more than one cell type due to an error in mitosis during development. Somatic mosaicism affects somatic tissues, sometimes only part of the body. Germline mosaicism shows normal parents but multiple affected offspring due to mutation in germline.

Relevant to dominant diseases: Expressivity – the amount that a trait is seen in an affected individual. “Variable expressivity” refers to members of the same family showing striking variation in phenotype, even with same gene. Thought to depend on environmental factors. Penetrance – The frequency a specific genotype is expressed by those individuals that posses it, usually given as a percentage. With low penetrance, some genetically affected individuals may show no evidence of trait. Age-related penetrance – refers to delayed age-of-onset, may be due to slow accumulation of toxins or inability to repair damage. NOTE: Penetrance is all-or-none, either the person does or does not express the disease gene. Expressivity refers to varying levels of severity of symptoms. Genetic anticipation – some dominant conditions become more sever and earlier-onset with successive generations New mutations – sporadic and rare, usually dominant, should consider non-paternity and reduced penetrance

Using a Punnett square: A a A AA Aa a Aa aa Chance of affected child = ¼ , chance of carrier child = ½ One parent recessive, one homozygous dominant Both parents heterozygous:

a a A Aa Aa A Aa Aa Chance of affected child = 0, chance of carrier child = 1

How to read pedigrees: Square is male (usually on left), circle is female, diamond is unspecified sex Black is affected, white is unaffected, dot in center is carrier, odd pattern of black (like one corner black) is affected and denotes different types of affected phenotypes. Arrow is proband – family member first brought to attention of clinician, often the person who sought medical advice. Not necessarily affected. Single lines show family tree relationships, diagonal line through symbol is deceased Double horizontal line is consanguineous mating by two related individuals. Be extra on the lookout for recessive traits appearing in their offspring! Draw oldest siblings on left, youngest on right

Genetics 4: Cancer Genetics and Genetic Counseling 8/23/11 I’m sorry if this is disjointed, these were weird learning objectives to write. 1) Define genetic counseling and integration into the clinical setting. Genetic Counseling: the process of helping people understand and adapt to the medical, psychological and familial implications of genetics’ effect on disease. i) Interpretation of family and medical history to assess chance of disease/ reoccurrences ii) Education about inheritance, testing, management, prevention, resources, research iii) Counseling to promote informed choices about risks The Process: a) Determine patient’s understanding b) Gather family history c) Identify syndromes in family history d) Determine if testing is reasonable e) Discuss risks, benefits and limitations of testing f) Interpret test g) define risk and make recommendations h) provide counseling, updates on information i) manage psychosocial impact on family 2) Summarize the basics of cancer genetics and clinical contribution of hereditary cancer syndromes. - All Cancer is genetic: - 10% hereditary - 20% is shared familial risk factors Genetic (inherited) vs Somatic (developed/ acquired) Gatekeeper Genes: act directly to control cellular proliferation / differentiation Tumor Suppressor Genes: negative effect on cell cycle regulation - cancers occur when both TSGs fail - like breaks on a car Proto-oncogenes: turn on genes that would normally be turned off - oncogne: mutated gene --> cancerous cell proliferation Caretaker Genes: genes that maintain integrity of genome DNA damage-response genes: repair mech for DNA during replication or from carcinogens. - cancer arises when both genes fail

3) Discuss the clinical impact of carrying a deleterious mutation in one of the BRCA genes. Both copies of a BRCA gene must be defective to get cancer (Knudson’s Two-Hit Hypothesis). If one gene is defective, the odds of acquiring a second hit are higher than with Sporadic (2 acquired mutations) cancer. Breast Cancer will be develop at a higher rate and earlier in life for this population. This population needs to: - Increase Surveillance (check for lumps/ mammograms/ MRI after 25) - Surveillance starting younger - Prevent Cancer through good lifestyle choices

4) Summarize the psychosocial considerations relevant to approaching the decision to undergo genetic testing for a hereditary cancer syndrome. a) Personal implications b) Familial implications c) Societal Implications - Very emotional time - impact on family - What if... positive? Negative? - Who to tell? Why or why not? - Family or cultural beliefs about cause of cancer - Risk for depression and/or guilt Other Terms: Synthetic Lethality: when mutations in either of two genes individually has no effect, but the combined mutations leads to cell death

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