statistics

Types of studies
Experimental;
- Individuals are divided into groups; 1 group taking a new drug & the other group take old drug or placebo (control gr). Which is either; 1. Double blind (doct & pt). 2. Single blind (pt). 3. Unblinded.

Crossover;
Each pt receive ttt & placebo, one following the other. Use small no of patients. Used in chronic dis & ttt that lead to temporary relief.

1.

Observational;
Only observe what happens without intervention. Cohort prospective; follow up study. e.g. take 1 group of smokers & non smokers & f.u. who will get the cancer. Case control; search in the past e.g. take cancer pt & search who was smoker in the past. (healthy control). Cross sectional; e.g. present pt on pills, how much have endometrial cancer?

2.
3.

Types of studies Cohort (prospective) study; one group with an exposure of interest is selected and
compared over time with another cohort without that exposure. to study short period disease e.g. smokers & follow them, use incidence. Disadv; expensive, liable to losses, drop out. Adv; 1. ideal to study disease causal relationship & temporal relationship between disease & risk exposure, 2. recall bias. 3. To see multiple outcomes. Cross sectional study; use prevalence to study the prevalence of disease. also called prevalence studies, look at the number of cases of a disease at a particular point in time. They are not useful for investigating rare diseases or exposures. Case control (retrospective) study; Adv; 1. To study rare diseases 2. Suitable for dis of long latent period 3. Less costly , easy, rapid. Disadv; 1. More prone to bias. 2. Difficult to prove temporal relationship between dis & exposure (risk)

 Meta analysis; when you combine data of 2 or more large previous studies to conclude new database regard the point of research. Advantage; rapid, cheap. Sequential trial; data are analyzed after each participant individual results become available, so the trial is continued until clear benefit is seen in one of comparing groups. Advantage ;shorter than fixed length trial, used when outcome of interest is known relatively quickly. factorial trials test two or more treatments simultaneously.

Ecological study; (Geographical studies) does not give data on individuals but give idea about average values on groups of people . (use prevalence or incidence) e.g. incidence of Ca colon in certain population. Screening test; 1. Should identify pts who require further investigations or ttt. 2. Should be safe, acceptable.

Q; A researcher is trying to design a study to find out the cause (or causes) of a rare disease, about which very little is known. What study design is most likely to be appropriate? A : Geographical B : Cross-sectional C : Cohort D : Intervention E : Case control.

Concerning cohort studies, which one of the following statements is true? A : They can only be used to compare two groups with one another. B : They are particularly useful with rare outcomes. C : Cohort studies are retrospective. D : They are better than other study designs for measuring prevalence of a disease in a population. E : They are better than other study types for measuring the incidence of a disease in a population.

Regarding case-control studies, which one of the following statements is FALSE? A : They are good for investigating rare diseases. B : They may be un-interpretable if controls are selected poorly. C : They are good for identifying rare causes of disease. D : They can examine multiple risk-factors for a single disease. E : They compare exposures of interest in cases and controls.

 Case-control studies compare exposures of interest in cases and controls. Two of their great strengths is that they can be used with rare diseases (because cases are pre-selected), and can examine multiple risk-factors (exposures). They are not good at identifying rare exposures. If the question is whether or not a rare exposure causes a disease then the appropriate design is a cohort study, where one group with the particular exposure of interest is compared with a control group without that exposure. The greatest difficulty in designing case-control studies is selection of an appropriate control group, and poor control selection often makes otherwise well-conducted studies uninterpretable.

Regarding crossover trial design, which one of the following statements is true? A : It can be used to compare treatments for an acute infection. B : It is a good method for comparing analgesics in arthritis. C : It cannot be double-blinded. D : It cannot be randomized. E : Tends to need more patients than are required with other trial designs to get adequate statistical power.

 B: The principle of a crossover design is that a patient has one drug or treatment, then a washout period, and then another drug, and the effect is compared between the two in a single individual. For this reason it is a good study design for treatment of chronic conditions, but not appropriate for acute conditions. It is just as easy (or difficult) to randomize and double-blind as for other study designs. Because each person is acting as their own control, it is usually possible to use smaller numbers to get the same power.

Interpreting Randomized controlled trials of therapeutic interventions (= experimental studies)

Types of errors; 1. Confounding; in which a measured effect attributed to a particular variable is in fact dt an unmeasured co- variable. e.g. age is a Confounding factor in a study of effect of folic acid supp. During pregnancy on neural tube defects. i.e. older women will have risk of neural tube defect irrespective of folic acid supp. Avoided by matching individuals in the groups acc. To potential confounders. 2. Bias; -Systemic differences bet groups that distorts the comparisons between those groups = non randomized sampling. Bias means a flaw in study design that leads to a built-in likelihood that the wrong result may be obtained. E.g. TB in Cairo. Avoided by random sampling & choose a reasonable study. 3. Sampling error; Arise because not all the population is examined but only a sample is taken. So the bigger the size of the sample, the smaller the sample error.

Types of data; 1. Qualitative data; proportion in the population 2. Quantitative data; mean, mode, median & standard deviation. Mean= is the arithmetic average.(sum/ n) Median= is the middle value.(1st + last /2) Mode= is the value that occurs most often.
mode median

Distribution;
100

mean
80 70 60 50

90 80 70

East 40

60 50 East Line 2 Line 3

30 20 10 0 1st Qtr

40 30 20 10

2nd Qtr

3rd Qtr

4th Qtr

5th

0 1st Qtr

2nd Qtr

3rd Qtr

4th Qtr

5th

Skewed distribution = asymmetrical

Normal distribution

Mean
Mean= X = (sum of all observed values) (n of sample size) It is a measure of the overall magnitude of the observations.

Standard deviation
- It is a measure of the variability of the values -is a measure of the scatter of observations about the mean - is the square of the variance SD= variance . - If the values are normally distributed, then: Approximately 68% of the values lie within +/- 1 SD of the mean. Approximately 95% of the values lie within +/- 2 SD of the mean. i.e. x +/- 2 SD should include about 95% of the observation.

 Mode is the value that has the largest frequency distribution (most frequent value). The median is the value above & below which of the values lie. () In the normal distribution the mean, mode, median all have the same value ( so we use the mean) but in the Skewed distribution, we prefer the median.

Q: If data are skewed, then they should be summarized in the form: A : mean and standard deviation. B : median and range C : mean and range D : median and standard deviation E : mean and 95% confidence intervals.

 B: Comment : Skewed data should always be summarized using the median and range. Standard deviation is based on the mean, which is not appropriate for skewed data. How do you decide if data are skewed? Plot them out and look at them, or find the median and calculate the mean: if these are more than slightly different, then the data is skewed.

Q: If a characteristic is normally distributed in a population; A this means that most of the population is composed of normal individuals B there will be equal numbers who have more or less of the characteristic than the mean C the median value will be greater than the mean D ten percent of individuals will be beyond two standard deviations from the mean .

 answer true = b a- nonsense b- i.e. the values will be symmetrical about the mean c- median =mean in normal distribution d-about 5% mode = mean

 The standard error is a measure of how precisely the sample mean approximates the population mean. standard error = standard deviation n as n , standard error .

Confidence intervals The interval (mean +/- 2 st error) is an approximate 95% confidence interval for the pop. mean i.e. we are 95% confident that the true mean lies inside this interval. The interval (mean +/- 1.64 st error) is a 90% confidence interval. NB; SD gives a measure of the spread of the data values. S error is a measure of how precisely the sample mean approximates the pop. Mean. E.g. FEV1 in 100 students Mean = 4.5 liter SD = 0.5 liters. - The interval where 95% of values lie is = mean +/- 2 st dev. =4.5 +/- 1 =3.5- 5.5 - The 95% confidence interval for pop mean= mean +/2 SE = 4.5 +/- 2 (0.5/ 100) = 4.5 +/- 2 (0.05)= 4.5 +/- 0.1= 4.4- 4.6 liters.

NB: the larger the sample size, the narrower the confidence interval. An outcome which varies widely in the pop will produce a wider confidence interval.

Correlation & regression

It is the relationship between 2 variables in the same individual (X & Y). r= correlation coefficient, r is never <-1 or >1, if r= +/- 1=perfect+/-=all pairs lie on the line, values near 1 or -1 show significant correlation. b= regression coefficient= measure the average increase in y/ unit increase in X= slope of the line.

0<r<1 B +ve

-1<r<0 B -ve

+ve correlation R=0 B=0

-ve correlation

R=0 B=0

No association

Non linear association

Q: The following statements are correct: A A correlation coefficient (r) of 0.04 is almost certainly significant. B The values of chi-squared range from 0 to +1. C The standard error of the mean is always less than the standard deviation. D A p value of 0.001 for the difference between two sample means is more significant than one of 0.01 E A confidence interval is the mean +/- 2 standard deviations.

 answer true = d a- values near 1 or -1 show significant correlation. b-probability (p) ranges from 0 to +1, c- standard error of the mean = standard deviation / square root of n. e-for a normal distribution a confidence interval for a mean can be given by +/1.96 standard errors of the mean.

How large was the difference between the intervention & the control groups in Randomized control trials
Control event rate (CER) = absolute risk. = risk of outcome (e.g. mortality) in the control group Experimental event rate (EER) = risk of outcome (e.g. mortality) in the Experimental gr .relative risk of avoiding the event= 100-EER. Absolute risk reduction (ARR) = CER-EER Relative risk reduction (RRR) = CER-EER/CER Number needed to treat (NNT)= 1/ARR

If 9.4% of patients given aspirin after myocardial infarction die (EER), compared with 11.8% of those not given aspirin (CER), then the absolute risk reduction (ARR) produced by aspirin is 11.8 9.4 = 2.4%, the relative risk reduction (RRR) when taking aspirin is 2.4/11.8 = 0.2 (20%), and the Number needed to treat (NNT) is 1 divided by 0.024 = 42, meaning that 42 patients with myocardial infarction must be treated with aspirin to prevent one death.

 the relative risk of avoiding the event (e.g.mortality) in experimental group = 100- EER= 100- 9.4=90.6%. The odds of avoiding the event (mortality) in experimental group = RR of avoiding / EER = 90.6/9.4=
the relative risk of avoiding the event (e.g.mortality) in the control group =100- 11.8= 88.2% . The odds of avoiding the event (e.g.mortality) in the control group =RR of avoiding /CER= 88.2 / 11.8 = . Odd ratio (OR)= odds of E/C

NB: In general, ORs & RRs tend to be rather similar when the CER < 20%, above this point, the figures divert (OR tends to be higher).

Q: A placebo-controlled study randomized 10 000 patients undergoing surgery for hip fracture to 160 mg aspirin / day, started preoperatively and continued for 35 days. About 1.5% of the patients allocated aspirin had DVT or pulmonary embolism (PE), compared with about 2.5% allocated placebo. Which one of the following statements about this trial is true? A : Aspirin produced a 1.5% absolute risk reduction in DVT/PE. B : Aspirin produced a 40% absolute risk reduction in DVT/PE. C : Aspirin produced a 1% proportional risk reduction in DVT/PE. D : Aspirin produced a 40% proportional risk reduction in DVT/PE. E : The Number Needed to Treat (NNT) to prevent one DVT/PE is 100/1.5 = 67.

 D: Comment : In this study aspirin reduces the risk of DVT/PE from 2.5% to 1.5%: this is an absolute risk reduction of 1% and a proportional (or relative) risk reduction of 1/2.5 = 40%. The NNT to prevent one DVT/PE is 1/absolute risk reduction = 1/0.01 = 100.

Q: In a study of patients with myocardial infarction, the death rate of those given aspirin is 8%,compared with 10% in those not given aspirin. This means that: A : the relative risk of death after myocardial infarction is 1.25 in those given aspirin B : the relative risk reduction produced by aspirin is 2% C : the number needed to treat with aspirin to prevent one death is 8 D : the number needed to treat with aspirin to prevent one death is 10 E : the absolute risk reduction produced by aspirin is 2%.

 Absolute risk reduction (or increase) = (Risk in group 1) minus (Risk in group 2), which is 2% in this example. Relative risk reduction is the difference of outcome in one group compared to another = (Risk in group 1) divided by (Risk in group 2). In this case aspirin reduced relative risk by 20%. The Number Needed to Treat = 1 divided by (Absolute Risk Reduction), which is 1/0.02 or 50 in this example.

 Probability (P) = Prevalence= % = diseased/population.

To convert probability to odd;

Odd= P/1-P.
e.g. 20% probability= 20/1-0.2 or 20/100-20= 20/80= 1/4= 0.25.

To convert odd to probability:

P= O/1+O. e.g. if odd = 1:3, prob= 1/1+3=1/4=25%. Means that for every 3 healthy there is 1 diseased so there is 1 dis among total of 4 so the diseased are 25%.

 P value is the probability of no difference (similarity) (null hypothesis). The smaller the P value, the bigger the difference. ( the more significant the difference) less likely of null hypothesis is true. If P value >0.05 the results would be found by chance in more than 1:20. The conventional cut-off for significance is P=0.05, or a 1-in-20 chance. Hence if 20 trials were conducted, you would expect to get one that was positive by chance alone. null hypothesis= non significant difference. Type I error; reject the null hypothesis while it is actually true. Help to determine sample size, In practice this means that the study claims to find a difference that does not really exist. Type II error; acceptance of the null hypothesis while it is false. Power of the study= 1- Type II error

 Sensitivity is the probability that a test will be positive when a patient has the condition. Sensitivity= true +ve /all diseased.
Specificity is the probability that a test will be negative when a patient does not have the condition. Specificity = true -ve /all non diseased (healthy). Positive predictive value of a test (PPP)= post test probability of a +ve test = true+ve/ all +ves. negative predictive value of a test (NPP)= post test probability of a -ve test = true-ve/ all -ves.
Diseased New test +ve True +ve non False +ve

New test -ve

False -ve

True -ve

Q: The specificity of a test is defined as follows:

A : The number of true negatives detected by the test divided by the number of all true negatives& false positive in the population tested.
B : The number of true positives detected by the test divided by the number of all true positives in the population tested. C : The number of true positives detected by the test divided by the total number of true positives in the population tested. D : The number of true negatives detected by the test divided by the total number of true negatives in the population tested. E : The number of true negatives detected by the test divided by the total number of true positives in the population tested.

 Likelihood ratio of a +ve test (LR+ve)= Sensitivity /1-specificity. Likelihood ratio of a -ve test (LR-ve)= 1-Sensitivity /specificity Pretest probability= prevalence in the population= diseased/ total no. Pretest odds= convert probability above to odds (Odd= P/1-P). Postest odds= Pretest odds X LR+ve Postest probability= convert above odds to probability (Postest P= O/1+O). Accuracy= all true results/ all results = probability of correct results. reliability is the ability of a test to produce the same result when repeated under identical conditions. The validity of the test is defined as the relevance of the test to the activities being treated. Efficacy= the effect of something under ideal or lab conditions.

 A clinical investigation examined the effectiveness of a new test in diagnosing Pancreatic carcinoma. The sensitivity was reported as 70%. Which one of the following statements is correct? 1 )70% of people will be correctly classified as having the disease 2 )70% of people with an abnormal test result will have the disease 3 )70% of people with a + test result will not have the disease 4 )70% of people with the disease will have an abnormal test result 5 )70% of people with the disease will have a - test result

Q: The statistical reviewer of a paper states

that they are concerned that the findings are biased. In statistical terms, bias means:
A : There is a flaw in study design that leads to a builtin likelihood that the wrong result may be obtained. B : There is a flaw in statistical analysis leading to a likelihood that the wrong result may be obtained. C : There is reason to believe that the authors wanted to obtain the result that the study showed. D : Both study design and statistical analysis are flawed, leading to a likelihood that the wrong result may be obtained. E : The study is not of sufficient statistical power to exclude the missing of a significant effect.

A Bias means a flaw in study design that leads to a built-in likelihood that the wrong result may be obtained. It cannot be controlled for at the analysis stage. It can be extremely difficult to design studies without potential bias, particularly when there are complex interactions between exposures under study. Techniques such as restriction and stratification are commonly used to reduce potential for bias.

A 95% confidence interval means: A : 95% of the data fall within the confidence interval. B : there is a 95% chance that two groups are different C : that p=0.05 D : there is a 95% chance that the true value falls within the confidence interval E : there is a 95% chance that the finding is clinically significant.

 D: Comment : The 95% confidence intervals (95% CI) around a value are the range within which there is a 95% chance that the true value lies. Similarly, the 95% CIs around a difference are the range in which there is a 95% chance that the true difference lies. If the means of two groups have overlapping 95% CIs, then the two groups are not statistically significantly different. If the 95% CI of the difference between two groups overlaps zero, then the difference between the two groups in not statistically significant. Statistical and clinical significance should not be confused. A very large study can generate very narrow 95% CIs (or very small p values) for very small differences, which may be of no clinical significance at all. By contrast, a small study may fail to show a statistically significant effect even if the effect is both large and clinically important.

Parametric tests means that the data will be of normal distribution. 1. Paired test or student T test is used if the members of the groups are well matched = paired. E.g. each diseased pt is matched with healthy individual of same age & sex. 2. Unpaired T test is used to compare the average values of 2 independent groups e.g. pts with & without disease/ treated vs. placebo. When you compare independent or diff groups, use unpaired t test.
Non- Parametric tests means that the data will be of Skewed distribution. Wilcoxon or Mann-Whitney U-test Chi-square test Used to compare % or proportion between 2 groups. In correlation coeffecient (r) r takes values between -1 & 1. the closer it is to zero, the less linear association.

1. 2.

 Chi-squared tests are used to compare 2 (%) or 2 proportions= used to test the difference between 2 nominal variables (count data). The larger the value X2 the smaller the p value, the more significant the test. Chi 2= X2= (observed-expected)2/expected to be compared with standard critical or standard table for determined degree of freedom. degree of freedom= (raws-1)x(columns-1). It assume that all table cells have expected freedum >1. It assume that 80% of table cells have expected freedum >5.

Regarding the description and comparison of two groups of data, which one of the following statements is true? A : Categorical data should be described as percentages and compared using a Students t-test. B : Normally distributed continuous data should be described as median and range and compared using a Chisquared test. C : Skewed continuous data should be described as median and range and compared using a Wilcoxon ranksum test. D : Normally distributed continuous data should be described as mean and standard deviation and compared using a Chi-squared test. E : Skewed continuous data should be described as mean and standard deviation and compared using a Students ttest.

 C Comment : Categorical variables are not continuous, e.g. drug / placebo, dead / alive. They should be described as percentages or proportions and compared with a Chisquared test. Normally distributed continuous data should be described as mean and standard deviation and compared with a Students t-test. Skewed continuous data should be described as median and range and compared using a test such as the Wilcoxon rank-sum test or the Mann-Whitney U-test.

 researcher compared the mean scores of nausea on a rating scale between standard therapy & a new drug in the treatment of chemotherapy induced nausea. Which one of the following is the appropriate statistical test? 1 )Chi-square test 2 )Paired T-test 3 )Life table analysis (log rank test) 4 )Pearson correlation 5 )Unpaired T-test

 Answer 5: The two-sample unpaired t test is used to test null hypothesis in two populations corresponding to two random samples are equal. For a paired t test, the data are dependent, i.e. there is a one-to-one correspondence between values in the two samples. For example,the same subject measured before & after a process change, & the same subject measured at different times.

Q: To compare two groups of categorical data, e.g. dead / alive by drug / placebo, the correct test is: A : Students t-test B : Analysis of variance (ANOVA) C : Wilcoxon rank-sum D : chi-squared E : p value

 D: Comment : Chi-squared tests (and variants there of) are widely used to compare percentages or proportions of categorical data. From the chi-squared statistic a p value is read off a statistical table to give the degree of significance. Traditionally a p value of less than 0.05, indicating a less than 5% probability that a result has arisen by chance, is taken (arbitrarily) as indicating that chance alone is not responsible for the difference between groups. Normally distributed data can be compared with a Students t-test (with correction for multiple comparisons when appropriate). Skewed continuous data can be compared with a Wilcoxon rank-sum test or a Mann-Whitney U-test.

In a trial of a new drug the following results were obtained:treatment group 44 improved 16 not improved, placebo group 36 improved 26 not improved. A the results so obviously show the benefit of treatment that statistical analysis is not required. B the data could be evaluated using the chi-squared test C Pearson's coefficient of linear regression would be an appropriate significance test D the numbers are too small to draw any conclusions E a Student t-test could be used

 answer true = b a-Nothing is ever that obvious surely. b-This data would be ideal for a chi-squared test. c-nonsense there is no linear regression to plot e-We are comparing proportions not means.

Q: Concerning the statistical power of studies, which one of the following statements is FALSE? A : International journals do not publish studies that are underpowered. B : A power calculation must always be performed before conducting randomized clinical trials. C : A type II error occurs if it is claimed two treatments are the same when the study is not large enough to detect equivalence. D : A type I error is where the null hypothesis is falsely rejected. E : The smaller the difference you want to detect, the larger a study must be.

 A: Comment : It is only ethical to conduct a clinical trial if it is capable of detecting a meaningful difference between two treatments to guide future practice. If a trial is underpowered it cannot detect a statistically significant difference. It is therefore mandatory to do a proper power calculation before exposing patients to a clinical trial. The rather daunting formal definition of a type I error means that a study falsely (but not deliberately) appears to find a difference between two groups which has actually arisen by chance alone. The conventional cut-off of p <0.05 will arise by chance alone one time in twenty. As many thousands of studies are published every month, type I errors are not rare. A type II error is formally where the null hypothesis is falsely accepted. To claim two treatments are equivalent requires huge numbers, and most studies are underpowered (too small) to reliably rule out a small difference between one treatment and another. Most published studies are small enough that type I and type II errors are a real possibility, and examples are published in good journals every week.

Q: A report of a clinical trial of a new analgesic states "In a comparison between the new drug and a placebo a higher proportion of patients taking the new drug obtained relief from pain (p<0.05)". It follows that:A the trial was well designed B amongst 100 patients treated with the drug five would be expected to have a placebo response C the result may have occurred by chance alone on less than one in 20 occasions D the probable error of the observations is +/- 5%

 answer true = c

Q: Regarding a randomized trial in which a new treatment for Clostridium difficile diarrhea is compared with an established treatment. A reviewer states that they are concerned that there might be type 2 statistical error. What does this mean? A : That the method of statistical analysis used is inappropriate. B : That the study has shown a difference between the treatments that is statistically significant but which is unlikely to be clinically significant C : That the study claims to find a difference that does not really exist, ie. the result is a statisticalfluke D : That the data is skewed (not normally distributed) and analysis should have used non-parametric rather than parametric statistical techniques E : That the study claims that there is no difference between the treatments, when in reality the trial was just too small to detect a difference.

 E: The null hypothesis is always that there is no difference between groups under study. A type 1 error occurs when the null hypothesis is falsely rejected. In practice this means that the study claims to find a difference that does not really exist,. A type 2 error occurs when the null hypothesis is falsely accepted. This means that it is claimed that there is no difference between two groups, when in reality the study is simply too small to detect a difference. This type of error can be avoided by making explicit power calculations before embarking on any study. This will answer the question if I am studying an outcome that occurs in (say) 20% of a conventionally treated group and want to show a (say) halving in the rate of this outcome, then how many patients do I need to study?

A type 1 statistical error in a clinical trial means that: A : patients were not allocated into groups with an appropriate randomisation method B : the null hypothesis is falsely accepted C : the null hypothesis is falsely rejected D : the statistical analysis was incomplete or incorrect E : the statement of the hypothesis to be tested was incomplete or flawed.

 C: Comment : A type 1 error is formally defined as being where the null hypothesis (which is that there is no difference between the groups) was falsely rejected. In practice this means that the study claims to find a difference that does not really exist.

Molecular medicine

Genome= complete complement of coding genes. Transcriptome= complete complement of expressed m RNA. Proteome= protein translated from m RNA. 2 organism may have the same n of genes but diff proteins dt; 1. Variation in gene expression (temporal, spatial). 2. Post transcription (diff. exons, splicing). 3. Post translation (glycosylation, sialydation).

 Human genome = 30.000 gene. Each cell express 16.000 gene. Housekeeping genes =genes expressed in all cells to provide basic function for cell survival (constitutive). Microarray analysis of transcriptome identify the expressed genes.e.g.

Gene
5
RNA polymerase binding site ex in ex in 3

Transforming factor binding sites

TATA box

ATG Translation Starting site

Translation termination code

Promotor elements

Exons= segment of the gene transcripted into m RNA then translated into proteins. Introns= segment of the gene transcripted then removed by splicing (not translated). Promotor elements = binding sites for initiation of transcription complex at 5. TF can activate any gene that has a TATA box. TATA box= - a promotor element. - at 25-30 base pairs from the start of transcription. - anchor to RNA polymerase II. Enhancers= - present at 5 or 3. - not obligatory for initiation. - but gene expression. TF; - basal = constitutive - Housekeeping genes. - inducible = temporal, spatial expression of genes for tissue phenotype. Application of TF; 1- many cong malformation are dt inherited mutation of TF. 2- can be oncogenic e.g. CMyC, P53. 3- steroids affect TF.

 cyclins= are proteins key regulators of cell cycles. telomere = DNA sequence at the end of each chromosome become progressively shorter with each cell division when it is reduced to a critical length, the cell is not capable of dividing. The enzyme telomerase lengthen it.

 Haplotype patterns= group of genes or alleles carried on the same chr, closely linked, travel together during meiosis, inherited as a unit. Gene mutations; - mismatch = change in the nucleotide. - inversion= nucleotide base removed, reverse directed & reinserted. - point mutation; single base pair substitution.

 Analysis of the proteome is better as it detects changes at the protein level, not reflected at transcriptome level dt Post translation processing.(bioinformatics).

 Direct DNA testing = identify abn within specific gene. - PCR. - restrictive enzyme digestion. - southern blot. - sequencing. Indirect DNA testing = unknown gene by tracking DNA markers in different members of the family (linkage analysis).

 southern blot (lab procedure); electrophoresis of DNA fragment through gel solid memb as nitrocellulose+ labelled probe visualised under x ray film. Northern blot is a mean to detect RNA (uracil instead of thymine in m RNA). Somatic cell hybridization; - method for gene maping. - using 2 diff species, chr from 1 species is selectively lost resulting in clones of certain chr of the another species. FISH; fluorescence in situ hybridization, labeled probes are hybridized to chromosomes, and the hybridized probes are detected with fluorochromes. visualised under florescent microscope, This technique is a rapid and sensitive means of detecting recurring numerical and structural abnormalities. for microdeletions & trisomy. SSCP (single strand conformation polymorphism analysis); is a technique for detecting variation in DNA sequence by running single stranded DNA fragments through a non denaturating gel.

 1. 2. 1. 2. 3. 4.

PCR Def; it is an amplification reaction in which a small amount of target DNA

(template) is amplified to produce enough amount to perform analysis. PCR is powerful (need only one copy). Uses= Detect viral or bact DNA. Detect mutations. Stages; Mix the specimen with 2 primers & Taq polymerase (thermostable DNA polymerase). Heat & cool primer anneal to template. Heat ( 72 c) polymerization. Repeat & analyse. Multiplex PCR; the use of more than 2 primers if more than one gene is to be identified. Nested PCR; when the sequence to be amplified need special definition e.g. resemble others. rt PCR (reverse transcriptase ); instead of DNA template, we take the m RNA (expressed genes) & transform it into DNA by reverse transcriptase enzyme (retroviral) as the m RNA is unstable. uses; - detection of expressed genes in tumor cells. - research; function of certain dis gene in diff tissues.

Preparation of monoclonal Ab;

For specific protein detection. 1. Inject Ag into an animal. 2. Hybridize its splenic cells + Myeloma cells (no longer produce its Ab) Ab to this Ag. 3. Select most specific Ab tissue culture. Uses; - diagnosis (scan) & TTT of cancer (drugs as majic bullet & radiotherapy). - Transplantation & immunomodulation (OKT3).

Receptors
A) Cell membrane surface receptors; 1- ligand gated Ion channel; e.g. neurotransmitter open ion channel - Nicotinic Na. - GABA, Glycine Cl. 2- receptors with protein tyrosine kinase (phosphorylation of tyrosine residue of receptors cascade of cytoplasmic prot). e.g. insulin, PDGF, prolactin, IGF1, MQ CSF, NGF, EGF. 3- G protein coupled ( is a protein that bind to guanine nucleotide). e.g. muscarinic, adrenergic. Dis associated with G protein abn e.g. cholera, Albright HOD, MeCune Albright S, pit adenoma. NB; prot kinases add phosphate group to serine, threonine, tyrosine residue (# phosphatase)

B) Nuclear H; - no 2 ry mess. - intracytoplasmic receptors. - the complex travel to the nucleus & bind to hormone responsive elements.

Molecular pathogenesis of cancer

1. Somatic evolution of Ca; escape from strictly regulated mechanism that control the growth of somatic cells. 2. Oncogenes; geneprotein cancer (=loss of growth control) protoncogene=Oncogene that is normally present in human cells i.e. C-onc e.g. C-Myc. Mutations resulting in tumors; RAS = G protein cell growth 1/3 of tumours if mutated. Mutations of protein kinasesTF (Fos & Jun)+ Myc tumor. In Burkitt lymphoma C Myc is transposed to Ig heavy chain locus on chr 14 in lymphocytes its expression & cellular mitosis. Philadelphia chr bcr + abl (9;22) fusion protein tumor growth. 3. Tumor suppressor gene; P53= normally function to inhibit cell cycle & abn growth, + apoptosis, if mutated tumor (most common cause of tumours). Le- Fraumeni S; AD dis, ccc by cancer breast, sarcoma, brain dt inactivation of P53. P27 Tumor suppressor gene through down regulation of cell cycle (cyclin dependent kinase inhibitor), if downregulated sporadic Cancer colon.

Apoptosis
Def; is the morphological changes that accompagny the programmed cell death e.g. cell shrinkage, compaction of chromatin, nuclear & cytoplasmic apoptotic bodies phagocytosed by MQ, laddering of DNA on electrophoresis gel by activation of intracellular nucleases. programmed cell death= naturally occurring cell death dt activation of a set of genes in response to ext signals e.g. from neighbour or extracellular matrix.

 Apoptosis; - non-inflammatory process. - no proteolytic enzymes. - no free radicals. - no damage of neighbouring cells. E.g. apopt of finger web, selection of neurons ( normal apopt in embrio). - apopt of excess or autoreactive T lymphocytes ( normal apopt in adult). - neurodegenerative dis, HIV (dis). - insufficient apoptosis e.g. cancer, autoimmune dis, viral dis. Factors that + apoptosis; P53, P27, Fas or CD95 (receptor for TNF), withdrawal of GF. Factors that - apoptosis; bcl2 (survival signals), B catenin accumulation adenoma. Apoptosis occur through proteases called caspases (e.g. ICE= IL-1B converting enzyme) that + endonuleases. Caspases = cysteine aspartate specific proteases.

P53 Fas, CD 95 +

+ _ Signals for cell death + bcl2

TF +

caspases

endonucleases

 Cancer resists apoptosis by; - Mutation of P53. - Causing apoptosis of cytotoxic T cells (TNF like + Fas). - Over-expression of Bcl2.

Nitric oxide (NO)

1. 2. Endothelial derived relaxation factor Produced from L arginine by oxidation of Nitrogen NO + citrulin. C GMP (2ry mess) in neighboring cells. Produced in; Constitutive..Vascular end & Nervous system VD, sm hyperplasia, plat agg. new memory. Inducible.. in MQ, PNL, plat, hepatocytecytotoxic.

Clinical application;
- So used as nitrates or inhaled NO in pulm HTN. - endothelial dysf in DM, HTN, smokers & hypercholestrolemia is dt loss of NO bioavailability. - NO in atherosclerosis, HTN dt CRF, HRS, Alzeheimer. - NO in septic shock, ARDS, acute inflammation.

Endothelin I (VC)
ET-1 (end, sm, coronary, GIT). Related to dis; - HTN, HRS, ARF, CHF, Raynaulds. (VC) - VC following subarachnoid Hge. ET1 receptor blockers & CEI used as anti HTN.

Pro-inflammatory cytokines
Il-1, TNF, TGF-B, Heat shock protein, free radicals. IL-1; Involved in; - Rh arthritis IL-1 + collagenase, phospholipase, cyclooxygenese (facilitator of damage). - Atherosclerosis; endo uptake of LDL IL-1 PDGF. - Septic shock IL1 NO, PG, PAF VD. - Infection, acute graft rejection IL1 T & B lymph.

 TNF; + GM- CSF, + PG. MQ, esinoph, NK. T lymph . dis associated; Rheumatoid, MS, MOF. So, neutralizing Ab = anti- TNF are used. not used in cancer highly toxic, + tumor growth.

TGF- action; - tissue repair. - extracellular matrix. - fibrosis. tissue injury + plat release of TGF-B chemotaxis monocytes (+ fibroblast GF, TNF, IL-1) involved in glomerulosclerosis, hep fibrosis, pulm fibrosis, bleomycin lung. NB;

HSPs (Heat shock proteins)

heat, chemicals, free radicals damage of intracellular proteins HSP cell resistance to stress through; - prot folding & unfolding. - degrad of prot ( by ubiquitination). dis associated; if mutated cataract, motor neuron deg. bact HSP + immune syst.

Free radicals Any molecule with 1or more unpaired electron (more reactive); peroxide, super, hydroxyl, NO. NB; hydroxyl is the most reactive. action; - lysosomes. - lipid peroxidation of memb. - mutations (by attaching purines & pyrimidine). Diseases athero, cancer, neurodeg ( MND). Free radical scavengers; - tocopherol (Vit E). - ascorbate (Vit C). - glutathione. - Beta carotene. - Flavenoids.

Adhesion Molecules
Def; Molecules that interact as receptors & ligand. 4 groups; Ig ( CD2, CD3, NCA (neural cell adh), ICAM (intercellular) bind to LFA ( lymph funct ass)to recruit lymphocytes. - integrin ( cell to matrix) Integrins are surface receptors by which cells are attached to extracellular matrix.. - Cadherins (Nerve & Muscle ). - selectins ( leukocytes to endoth in inflam, over expressed in autoimmune viral hepatitis, organ rejection). Clinical application; 1. leuk adhesions deficiency recc bact sepsis. 2. integrin IIb IIIa ( plat receptor to fibrinogen) deficiency Glansman thrombathenia. Ab (abciximab) antithrombotic in coronary Ht.

Stem cells
progenitor cells. present in certain tissues e.g. BM, embrionic. embryonic totipotent (any tissue). BM Bl. cells only, can be recruited by Ag sorting with CD34 Ab & undergo transdifferentiation to non hematologic cells.