Molecular diagnostics in Medicine

Question 1

Types of genetic tests (hint: screening to diagnose and predict prognosis)

Answer 1

“screening to diagnose and predict prognosis”

Screening Test

Diagnostic test (e.g. CF)

Predictive test (e.g. BRCA/cancer)

Prognostic test (cancers)

Question 2

Test characteristics (2 categories)

Answer 2

APSS (technical)

Accuracy

Precision

Sensitivity

Specificity

(clinical/patient related)

Clinical Validity

Clinical Utility

Personal Utility

Question 3

Clinical validity

Answer 3

Accuracy of disease prediction/how well test predicts disease

Question 4

Clinical utility

Answer 4

Usefulness of test results treatment/patient management

Question 5

Personal utility

Answer 5

Effect of test results on patient (patient’s life basically)

Question 6

Categories of etiologies

Answer 6

Point mutations

Copy number changes

Repeat expansions

Epigenetic changes

Question 7

Point mutation types

Answer 7

Frameshift

Missense (conservative vs nonconservative)

Nonsense

Silent

Question 8

Conservative vs Nonconservative missense mutations

Answer 8

Conservative - results in AA change; new AA resembles WT AA in function (so no functional difference)

Non-conservative - results in AA change; new AA functionally different from WT AA (gain or loss of function)

Question 9

Types of Copy number changes

Answer 9

Aneuploidy; Translocation

Microdeletion/duplication (e.g. CF = loss of 1 bp)

Gene gain/loss

Exon gain/loss

Question 10

Repeat expansion types and locations

Answer 10

Trinucleotide expansion (CGG) or Dinucleotide

Located within gene, promoter or intron

Question 11

Epigenetic changes

Answer 11

Methylation of CpG islands

Question 12

Molecular diagnostic techniques

“RFLP e sequence”

“Microsatellites methylate ASO’s”

Answer 12

RFLP

Sequencing

Microsatellite analysis

Methylation testing (via Southern)

Allele-specific oligonucleotide

RFLPing the sequence!!

Microsatellites methyl8 ASO’s!!

Question 13

Point mutation genetic test used

Answer 13

RFLP (SNP type)

Sequencing

Question 14

Test for repeat expansions

Answer 14

Microsatellite analysis

Question 15

Test for methylation

Answer 15

Southern Blot

Question 16

Test for copy number changes

Answer 16

Microarray

Multiplex ligation-dependent probe amplification

Question 17

Multiplex ligation dependent amplification

Answer 17

Essentially, PCR with 2 primers >>

each w/ probe on one end>>

If mutated sequence = amplification but wrong size

If no mutation = amplification + right size

Question 18

Next generation sequencing types

Answer 18

Targeted gene panels

Exome sequencing (highest coverage) [coding regions only]

(Whole) Genome sequencing

Question 19

3 false negative scenarios

Answer 19

Question 20

Sequence interpretation

Answer 20

Question 21

Down Syndrome etiologies

Answer 21

Question 22

Tests for DS

Answer 22

Question 23

Test to order for DS

Answer 23

Question 24

Fragile X Syndrome etiology

Answer 24

Question 25

Tests for Fragile X Syndrome

Answer 25

Question 26

Which test would you order for Fragile X?

Answer 26

Trinucleotide repeat analysis

Question 27

Intepret the Southern Blot below of Fragile X patients. Give reasons for each intepretation.

Answer 27

Question 28

Prader Willi Syndrome etiologies

Answer 28

Question 29

PWS testing

Answer 29

Question 30

Why would you choose methylation testing for PWS and not any of the other tests?

Answer 30

MLPA and FISH will only detect gain/loss of genetic info

Microarray won’t detect UPD

UPD analysis won’t detect deletion

Question 31

How do you interpret results of PWS detection using UPD analysis?

Answer 31

Look at peak size

Look at genes present (from both parents) in proband

Question 32

How do you interpret the results of PWS detection by Microarray?

Answer 32

As with any other microarray (discussed in this lecture), compare the patient’s genetic profile to that of the control

Question 33

Summary of disease examples

Answer 33