statistics & molecular mrcp1

7/27/2019 Statistics & Molecular MRCP1

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statistics


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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.

Observational; Only observe what happens without intervention.1. Cohort prospective; follow up study.

e.g. take 1 group of smokers & non smokers & f.u. who will get the cancer.2. Case control; search in the past

e.g. take cancer pt & search who was smoker in the past. (healthy control).

3. Cross sectional; e.g. present pt on pills, how much have endometrial cancer?


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Types of studiesCohort (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 calledprevalence studies, look at the number of cases of a disease at a particular pointin 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)


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Meta analysis; when you combine data of 2 or more largeprevious studies to conclude new database regard thepoint of research. Advantage; rapid, cheap.

Sequential trial; data are analyzed after each participantindividual results become available, so the trial iscontinued until clear benefit is seen in one of comparinggroups. Advantage ;shorter than fixed length trial, usedwhen outcome of interest is known relatively quickly.

factorial trials test two or more treatments simultaneously.


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Ecological study;(Geographical studies)does not give data on individuals but give

idea about average values on groups ofpeople . (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.


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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 : CohortD : Intervention

E : Case control.


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Concerning cohort studies, which one of the following

statements is true?

A : They can only be used to compare two groupswith 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 apopulation.


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Regarding case-control studies, which one of thefollowing statements is FALSE?

A : They are good for investigating rare diseases.

B : They may be un-interpretable if controls areselected poorly.

C : They are good for identifying rare causes ofdisease.

D : They can examine multiple risk-factors for a

single disease.E : They compare exposures of interest in cases

and controls.


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Case-control studies compare exposures of interest incases and controls. Two of their great strengths is thatthey can be used with rare diseases (because cases arepre-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 interestis compared with a control group without that exposure.

The greatest difficulty in designing case-control studiesis selection of an appropriate control group, and poorcontrol selection often makes otherwise well-conductedstudies uninterpretable.


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Regarding crossover trial design, which one of thefollowing 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 statisticalpower.


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B : The principle of a crossover design is that a

patient has one drug or treatment, then awashout period, and then another drug, and the

effect is compared between the two in a singleindividual.

For this reason it is a good study design fortreatment of chronic conditions, but not

appropriate for acute conditions. It is just aseasy (or difficult) to randomize and double-blindas for other study designs. Because eachperson is acting as their own control, it is usuallypossible to use smaller numbers to get the same

power.


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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. Duringpregnancy 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 thewrong 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.


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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.

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Normal distributionSkewed distribution= asymmetrical

meanmedian

mode Distribution;


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MeanMean= 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.


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Mode is the value that has the largestfrequency 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 themedian.


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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 deviationE : mean and 95% confidence intervals.


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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 theseare more than slightly different, then thedata is skewed.


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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 moreor less of the characteristic than the mean

C the median value will be greater than the

meanD ten percent of individuals will be beyond two

standard deviations from the mean .


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answer true = b

a- nonsense

b- i.e. the values will be symmetrical aboutthe mean

c- median =mean in normal distribution

d-about 5%mode = mean


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The standard error is a measure of howprecisely the sample mean approximates

the population mean.

standard error= standard deviation

nas n , standard error .


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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% confidenceinterval.NB;

SD gives a measure of the spread of the data values. S erroris 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 +/- 2st 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.


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NB:

the larger the sample size, the narrowerthe confidence interval.

An outcome which varies widely in the popwill produce a wider confidence interval.


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Correlation & regression It is the relationship between 2 variables in the same individual (X & Y). r= correlation coefficient, r is never 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/ unitincrease in X= slope of the line.

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Q: The following statements are correct:

A A correlation coefficient (r) of 0.04 is almostcertainly significant.

B The values of chi-squared range from 0 to +1.

C The standard error of the mean is always lessthan the standard deviation.

D A p value of 0.001 for the difference betweentwo sample means is more significant than one

of 0.01E A confidence interval is the mean +/- 2 standard

deviations.


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answer true = da- values near 1 or -1 show significant

correlation.

b-probability (p) ranges from 0 to +1,

c- standard error of the mean = standarddeviation / square root of n.

e-for a normal distribution a confidenceinterval for a mean can be given by +/-1.96 standard errors of the mean.


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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/CERNumber 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.


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the relative risk of avoiding the event (e.g.mortality) inexperimental group = 100- EER= 100- 9.4=90.6%.

The odds of avoiding the event (mortality) in experimental

group = RR ofavoiding / EER = 90.6/9.4=

the relative risk of avoiding the event (e.g.mortality) inthe control group =100- 11.8= 88.2% .

The odds of avoiding the event (e.g.mortality) in thecontrol group =RR ofavoiding /CER= 88.2 / 11.8 = .

Odd ratio (OR)= odds of E/C

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


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Q: A placebo-controlled study randomized 10 000 patientsundergoing 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 orpulmonary embolism (PE), compared with about 2.5%allocated placebo. Which one of the following statementsabout 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 inDVT/PE.

D : Aspirin produced a 40% proportional risk reduction inDVT/PE.

E : The Number Needed to Treat (NNT) to prevent oneDVT/PE is 100/1.5 = 67.


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D :

Comment : In this study aspirin reducesthe 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 is1/absolute risk reduction = 1/0.01 = 100.


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Q: In a study of patients with myocardial infarction, thedeath rate of those given aspirin is 8%,compared with10% in those not given aspirin. This means that:

A : the relative risk of death after myocardial infarction is

1.25 in those given aspirinB : the relative risk reduction produced by aspirin is 2%

C : the number needed to treat with aspirin to prevent onedeath is 8

D : the number needed to treat with aspirin to prevent onedeath is 10

E : the absolute risk reduction produced by aspirin is 2%.


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Absolute risk reduction (or increase) = (Risk ingroup 1) minus (Risk in group 2), which is 2% inthis example.

Relative risk reduction is the difference ofoutcome in one group compared to another =(Risk in group 1) divided by (Risk in group 2). Inthis case aspirin reduced relative risk by 20%.

The Number Needed to Treat = 1 divided by(Absolute Risk Reduction), which is 1/0.02 or 50in this example.


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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 sothere is 1 dis among total of 4 so the diseased are

25%.

P value is the probability of no difference (similarity) (null


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P value is the probability of no difference (similarity) (nullhypothesis).

The smaller the P value, the bigger the difference. ( the moresignificant the difference) less likely of null hypothesis is true.

IfP value >0.05 the results would be found by chance in morethan 1:20.

The conventional cut-off for significance is P=0.05, or a 1-in-20chance. Hence if 20 trials were conducted, you would expect toget 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


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Sensitivityis the probability that a test will be positivewhen a patient has the condition.

Sensitivity= true +ve /all diseased.

Specificityis the probability that a test will be negativewhen a patient does not have the condition.

Specificity = true -ve /all non diseased (healthy).

Positive predictive value of a test (PPP)= post testprobability 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.

nonDiseased

False +veTrue +veNew test +ve

True -veFalse -veNew test -ve


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

A : The number of true negatives detected by the test divided by thenumber 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 totalnumber of true positives in the population tested.

D : The number of true negatives detected by the test divided by the totalnumber of true negatives in the population tested.

E : The number of true negatives detected by the test divided by the totalnumber of true positives in the population tested.

Lik lih d ti f + t t (LR+ ) S iti it /1 ifi it


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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 correctresults.

reliabilityis the ability of a test to produce the same resultwhen repeated under identical conditions.

The validity of the test is defined as the relevance of the test tothe activities being treated.

Efficacy= the effect of something under ideal or lab conditions.


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A clinical investigation examined the effectiveness of anew test in diagnosing Pancreatic carcinoma. The

sensitivity was reported as 70%. Which one of thefollowing statements is correct?

1 )70% of people will be correctly classified as havingthe disease

2 )70% of people with an abnormal test result will havethe disease

3 )70% of people with a + test result will not have thedisease

4 )70% of people with the disease will have an abnormal

test result5 )70% of people with the disease will have a - test result


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Q: The statistical reviewer of a paper statesthat they are concerned that the findingsare biased. In statistical terms, bias

means:A : There is a flaw in study design that leads to a built-

in 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 resultmay be obtained.

E : The study is not of sufficient statistical power toexclude the missing of a significant effect.


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A

Bias means a flaw in study design that leads to abuilt-in likelihood that the wrong result may be

obtained. It cannot be controlled for at theanalysis stage. It can be extremely difficult to

design studies without potential bias, particularly

when there are complex interactions between

exposures under study. Techniques such asrestriction and stratification are commonly used

to reduce potential for bias.


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A 95% confidence interval means:

A : 95% of the data fall within the confidenceinterval.

B : there is a 95% chance that two groups aredifferent

C : that p=0.05

D : there is a 95% chance that the true valuefalls within the confidence interval

E : there is a 95% chance that the finding isclinically significant.


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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 twogroups are not statistically significantly different. If the 95% CI of thedifference 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 verylarge study can generate very narrow 95% CIs (or very small pvalues) for very small differences, which may be of no clinicalsignificance 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.


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Parametric tests means that the data will be ofnormal distribution.

1. Paired test or student T test is used if the members of the groups arewell matched = paired. E.g. each diseased pt is matched withhealthy individual of same age & sex.

2. Unpaired T test is used to compare the average values of 2independent 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 ofSkeweddistribution.1. Wilcoxon or Mann-Whitney U-test

2. Chi-square test Used to compare % or proportion between 2groups.

In correlation coeffecient (r) r takes values between -1 & 1. thecloser it is to zero, the less linear association.


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Chi-squared tests are used to compare 2 (%) or 2proportions= used to test the difference between 2 nominalvariables (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 standardtable for determined degreeof 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.

R di th d i ti d i f t


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Regarding the description and comparison of two groupsof data, which one of the following statements is true?

A : Categorical data should be described as percentagesand compared using a Students t-test.

B : Normally distributed continuous data should bedescribed as median and range and compared using a Chi-squared test.

C : Skewed continuous data should be described as

median and range and compared using a Wilcoxon rank-sum test.

D : Normally distributed continuous data should bedescribed as mean and standard deviation and comparedusing a Chi-squared test.

E : Skewed continuous data should be described as meanand standard deviation and compared using a Students t-test.


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C Comment : Categorical variables are not continuous, e.g.

drug / placebo, dead / alive. They should be describedas percentages or proportions and compared with a Chi-

squared test. Normally distributed continuous data should be

described as mean and standard deviation andcompared with a Students t-test.

Skewed continuous data should be described as medianand range and compared using a test such as theWilcoxon rank-sum test or the Mann-Whitney U-test.


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researcher compared the mean scores ofnausea on a rating scale between standardtherapy & a new drug in the treatment ofchemotherapy 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 correlation5 )Unpaired T-test


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Answer5: The two-sample unpaired t test is used to test

null hypothesis in two populationscorresponding to two random samples areequal.

For a paired t test, the data are dependent, i.e.there is a one-to-one correspondence betweenvalues in the two samples. For example,thesame subject measured before & after aprocess change, & the same subjectmeasured at different times.


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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


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D : Comment : Chi-squared tests (and variants there of) are

widely used to compare percentages or proportions ofcategorical data. From the chi-squared statistic a p valueis read off a statistical table to give the degree ofsignificance. 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 thatchance alone is not responsible for the differencebetween groups.

Normally distributed data can be compared with aStudents t-test (with correction for multiple comparisonswhen appropriate).

Skewed continuous data can be compared with aWilcoxon rank-sum test or a Mann-Whitney U-test.


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In a trial of a new drug the following results were obtained:-treatment group 44 improved 16 not improved, placebogroup 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 anappropriate significance test

D the numbers are too small to draw any conclusions

E a Student t-test could be used


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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.


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Q: Concerning the statistical power of studies, which one ofthe following statements is FALSE?

A : International journals do not publish studies that areunderpowered.

B : A power calculation must always be performed beforeconducting randomized clinical trials.

C : A type II error occurs if it is claimed two treatments arethe same when the study is not large enough to detectequivalence.

D : A type I error is where the null hypothesis is falselyrejected.

E : The smaller the difference you want to detect, the largera study must be.


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A : Comment : It is only ethical to conduct a clinical trial if it is capable of

detecting a meaningful difference between two treatments to guidefuture practice. If a trial is underpowered it cannot detect a

statistically significant difference. It is therefore mandatory to do a proper power calculation beforeexposing patients to a clinical trial. The rather daunting formaldefinition of a type I error means that a study falsely (but notdeliberately) appears to find a difference between two groups whichhas actually arisen by chance alone. The conventional cut-off of p


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Q: A report of a clinical trial of a new analgesicstates "In a comparison between the new drugand a placebo a higher proportion of patientstaking the new drug obtained relief from pain

(p


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answer true = c

Q: Regarding a randomized trial in which a new treatment for


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Q ega d g a a do ed a c a e ea e oClostridium difficile diarrhea is compared with an establishedtreatment. A reviewer states that they are concerned thatthere 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 unlikelyto be clinically significant

C : That the study claims to find a difference that does notreally exist, ie. the result is a statisticalfluke

D : That the data is skewed (not normally distributed) andanalysis should have used non-parametric rather thanparametric statistical techniques

E : That the study claims that there is no difference betweenthe treatments, when in reality the trial was just too small todetect a difference.


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E : The null hypothesis is always that there is no difference

between groups under study. A type 1 error occurs whenthe null hypothesis is falsely rejected. In practice thismeans that the study claims to find a difference that

does not really exist,. A type 2 error occurs when the nullhypothesis is falsely accepted. This means that it isclaimed that there is no difference between two groups,when in reality the study is simply too small to detect adifference. This type of error can be avoided by making

explicit power calculations before embarking on anystudy. This will answer the question if I am studying anoutcome that occurs in (say) 20% of a conventionallytreated group and want to show a (say) halving in therate of this outcome, then how many patients do I needto study?


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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 acceptedC : the null hypothesis is falsely rejected

D : the statistical analysis was incomplete or incorrect

E : the statement of the hypothesis to be tested wasincomplete or flawed.


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C :

Comment : A type 1 error is formallydefined as being where the null hypothesis

(which is that there is no differencebetween the groups) was falsely rejected.

In practice this means that the study

claims to find a difference that does notreally exist.


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Molecular medicine


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Genome= complete complement of codinggenes.

Transcriptome= complete complement ofexpressed m RNA.

Proteome= protein translated from m RNA.

2 organism may have the same n of genes butdiff proteins dt;1. Variation in gene expression (temporal,

spatial).

2. Post transcription (diff. exons, splicing).3. Post translation (glycosylation, sialydation).


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Human genome = 30.000 gene. Each cell express 16.000 gene. Housekeeping genes =genes expressed in

all cells to provide basic function for cellsurvival (constitutive).

Microarray analysis of transcriptomeidentify the expressed genes.e.g.


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Gene

Transforming factor

binding sitesTATA

box

RNA polymerase

binding site

ex exin in

ATG

Translation

Starting site

Translation

termination code

Promotor elements

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Exons= segment of the gene transcripted into m RNA then translated into


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Exons= segment of the gene transcripted into m RNA then translated intoproteins.

Introns= segment of the gene transcripted then removed by splicing (nottranslated).

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 or3.- 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.


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cyclins= are proteins key regulators of cellcycles.

telomere = DNA sequence at the end ofeach chromosome become progressivelyshorter with each cell division when it is

reduced to a critical length, the cell is not

capable of dividing. The enzymetelomerase lengthen it.


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Haplotype patterns= group of genes or allelescarried 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.


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Analysis of the proteome is better as itdetects changes at the protein level, not

reflected at transcriptome level dt Post

translation processing.(bioinformatics).


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Direct DNA testing = identify abn withinspecific gene.

- PCR.

- restrictive enzyme digestion.- southern blot.

- sequencing.

Indirect DNA testing = unknown gene bytracking DNA markers in differentmembers of the family (linkage analysis).

southern blot (lab procedure); electrophoresis ofDNAfragment through gel solid memb as nitrocellulose+


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fragment through gel solid memb as nitrocellulose+labelled probe visualised under x ray film.

Northern blot is a mean to detect RNA (uracil instead ofthymine in m RNA).

Somatic cell hybridization;- method for gene maping.

- using 2 diff species, chr from 1 species is selectively lostresulting in clones of certain chr of the another species.

FISH; fluorescence in situ hybridization, labeled probes arehybridized to chromosomes, and the hybridized probes aredetected with fluorochromes. visualised underflorescentmicroscope, This technique is a rapid and sensitive means of

detecting recurring numerical and structural abnormalities

.for

microdeletions & trisomy.

SSCP (single strand conformation polymorphism analysis); isa technique for detecting variation in DNA sequence byrunning single stranded DNA fragments through a nondenaturating gel.

PCR


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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=1. Detect viral or bact DNA.2. Detect mutations.

Stages;1. Mix the specimen with 2 primers & Taq polymerase (thermostable DNA

polymerase).

2. Heat & cool

primer anneal to template.3. Heat ( 72 c) polymerization.

4. Repeat & analyse.

Multiplex PCR; the use of more than 2 primers if more than one gene is tobe 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 mRNA (expressed genes) & transform it into DNA by reverse transcriptaseenzyme (retroviral) as the m RNA is unstable.

uses; - detection of expressed genes in tumor cells.

- research; function of certain dis gene in diff tissues.


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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 asmajic bullet & radiotherapy).

- Transplantation & immunomodulation (OKT3).

Receptors


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Receptors

A) Cell membrane surface receptors;

1- ligand gated Ion channel;e.g. neurotransmitteropen ion channel

- Nicotinic Na.

- GABA, Glycine Cl.

2- receptors with protein tyrosine kinase (phosphorylation oftyrosine 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)


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B) Nuclear H;

- no 2 ry mess.

- intracytoplasmic receptors.

- the complex travel to the nucleus & bind

to hormone responsive elements.

Molecular pathogenesis of cancer


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Molecular pathogenesis of cancer1. 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)+ Myctumor. In Burkitt lymphoma C Myc is transposed to Ig heavy chain locuson 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 dtinactivation of P53.

P27 Tumor suppressor gene through down regulation of cell cycle(cyclin dependent kinase inhibitor), if downregulated sporadicCancer colon.


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Apoptosis

Def; is the morphological changes that accompagnythe 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 toext signals e.g. from neighbour or extracellular

matrix.


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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-1Bconverting enzyme) that + endonuleases.

Caspases = cysteine aspartate specific proteases.


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Signals for cell death

+

+

+

P53

Fas, CD 95

TF

_bcl2

+

caspases

endonucleases

+


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Cancer resists apoptosis by;- Mutation of P53.

- Causing apoptosis of cytotoxic T cells

(TNF like + Fas).

- Over-expression of Bcl2.

Nitric oxide


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Nitric oxide

(NO)

Endothelial derived relaxation factor Produced from L arginine by oxidation of Nitrogen NO + citrulin. C GMP (2ry mess) in neighboring cells. Produced in;1. Constitutive..Vascular end & Nervous systemVD, sm

hyperplasia, plat agg. new memory.2. 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 dtloss of NO bioavailability.

- NO in atherosclerosis, HTN dt CRF, HRS, Alzeheimer.- NO in septic shock, ARDS, acute inflammation.

Endothelin I (VC)


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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


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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 IL1NO, PG, PAF VD.

- Infection, acute graft rejection IL1 T & Blymph.


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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, + tumorgrowth.

TGF


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TGF-

action;

- tissue repair.- extracellular matrix.- fibrosis.

tissue injury + plat release of TGF-Bchemotaxis monocytes (+ fibroblast GF,TNF, IL-1)

involved in glomerulosclerosis, hep fibrosis,pulm fibrosis, bleomycin lung. NB;

HSPs (Heat shock proteins)


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HSPs (Heat shock proteins)

heat, chemicals, free radicals damage of

intracellular proteins HSP cellresistance 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


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Any molecule with 1or more unpaired electron (morereactive); 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


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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 bywhich cells are attached to extracellular matrix..

- Cadherins (Nerve & Muscle ).- selectins ( leukocytes to endoth in inflam, over expressedin 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.

St ll


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Stem cells

progenitor cells. present in certain tissues e.g. BM, embrionic.

embryonic totipotent (any tissue).

BM Bl. cells only, can be recruited by Agsorting with CD34 Ab & undergo

transdifferentiation to non hematologic cells.

statistics & molecular mrcp1

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