Exam #3: Molecular Pathology

Question 1

What are four basic applications of molecular diagnosis?

Answer 1

1) Detect inherited or acquired mutations
2) Diagnose/classify of tumors
3) Detect infectious agents e.g. HIV…etc.
4) Tissue typing, paternity testing, and forensic medicine

Question 2

What is the definition of cytogenetic analysis?

Answer 2

Cytogenetic analysis or “Karyotyping” refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two

Question 3

What is cytogenetic analysis useful for detecting?

Answer 3

Large scale chromosomal abnormalities such as:

1) Aneuploidy e.g. Down’s Syndrome
2) Sex chromosome abnormalities e.g. Turner’s Syndrome
3) Fragile X Syndrome
4) Translocations

Question 4

What is molecular analysis?

Answer 4

Analysis of DNA & RNA

Question 5

What is molecular analysis used for?

Answer 5

Looking for subtle changes that cannot be detected by cytogenetics

Question 6

What types of mutations is cytogenetic analysis used for?

Answer 6

1) Genome mutations= loss or gain of an entire chromosome

2) Chromosome mutations= rearrangement of genetic material

Question 7

What type of mutations is molecular analysis used for?

Answer 7

Gene mutations= complete deletion of a gene to a single point mutation

Thus, molecular analysis is used to look for subtle changes that cannot be detected by cytogenetics

Question 8

What are the four different types of gene mutations

Answer 8

1) Point mutations
2) Insertions
3) Deletions
4) Trinucleotide repeat mutations

Question 9

What is a trinucleotide repeat mutation?

Answer 9

DNA contains regions with triplets; these are potentially unstable regions of DNA that are prone to expansion i.e. mutation

**The diseases caused by trinucleotide expansions are referred to as Trinucleotide Repeat Disorders

Question 10

List examples of diseases that are caused by trinucleotide repeat expansions.

Answer 10

• Myoclonus Epilepsy
• Fragile X Syndrome
• Friedreich Ataxia
• Huntington Disease
• Myotonic Dystrophy

Question 11

Where are the expansions for: Myoclonus Epilepsy, Fragile X Syndrome, Friedreich Ataxia, Huntington Disease, & Myotonic Dystrophy located in the gene?

Answer 11

• Myoclonus Epilepsy= Promoter
• Fragile X Syndrome= UTR
• Friedreich Ataxia= Intron
• Huntington Disease= Exon
• Myotonic Dystrophy= UTR

Question 12

What is pre-mRNA?

Answer 12

mRNA that has not be processed i.e. not spliced

Question 13

What causes errors in mRNA splicing?

Answer 13

Single point mutations in introns can cause splicing errors

Question 14

What is an example of a disease caused by a splice error of pre-mRNA?

Answer 14

Beta-thallassemia

Question 15

After splicing, how else is pre-mRNA processed?

Answer 15

1) 5’ cap

2) Poly-A tail

Question 16

What happens when there are mutations in the polyadenylation signal of mRNA processing?

Answer 16

Results in unstable mRNA

Question 17

Give an example of a disease caused by a mutation in polyadenylation signal.

Answer 17

Beta-thallassemia

I.e. B-tallasemia can be caused by a variety of mRNA processing mutations

Question 18

What is the difference between direct detection of mutations in DNA vs. indirect detection?

Answer 18

Direct
- Comparison of mutated sequence with known normal sequence

Indirect

• Sequence unknown
• Uses linkage markers

Question 19

Outline the process of direct detection of DNA mutations.

Answer 19

1) Amplify gene of interest with PCR
2) Digest PCR products w/ restriction enzyme (endonuclease)
3) Separate digested products w/ electrophoresis

*****Mutations are determined based off of size differences i.e. distance traveled on the gel

Question 20

Give an example of a disease that is detected with direct analysis.

Answer 20

Coagulation Factor V

Question 21

What is allele-specific extension?

Answer 21

This is also known as SNP genotyping, the measurement of genetic variations of single nucleotide polymorphisms (SNPs)

**This is a “direct detection” technique & that it can be done in real time so that the time of PCR amplification can be reduced

Question 22

Outline the process of allele-specific extension/ SNP genotyping.

Answer 22

1) Amplify segment of interest
2) Add complimentary florescent nucleotides–one for mutant & one for normal nucleotide
3) Different color for normal & mutant

*A mix of colors= heterozygous mutation

Question 23

Generally, how is indirect gene analysis performed?

Answer 23

Linkage of the disease gene with a marker that is nearby

Question 24

What does indirect linkage analysis require? What does it not require?

Answer 24

DOES NOT require knowledge of the EXACT mutation, but DOES require the polymorphism used for detection to be sufficiently close to the mutated gene so that the marker & mutation are inherited together

Question 25

Outline the process of linkage analysis/ indirect detection.

Answer 25

1) Gene mutation and marker are inherited together
2) Marker gene/ mutant are associated with a new “cutting site” when treated with an endonuclease
3) Endonuclease processing & southern blot–>different bands

Question 26

What is the mnemonic to remember what southern, northern, & western blots are analyzing?

Answer 26

SNoW
DRoP

Southern= DNA
Northern= RNA
Western= Protein

Question 27

What are the major three infectious agents that are detected with molecular analysis?

Answer 27

1) HIV
2) Hep B
3) Hep C

Question 28

How is the quantification of RNA used clinically?

Answer 28

Guide for therapy e.g. quantification of HIV RNA is an important guide for therapy

Question 29

How is molecular analysis used in forensic medicine?

Answer 29

1) PCR DNA samples w/ standard set of primers to amplify DNA fragments that are highly polymorphic i.e. MHC locus
2) Compare fragments to controls

Question 30

What is FISH?

Answer 30

Flourescence In Situ Hybridization

Question 31

How can FISH be used clincally?

Answer 31

Detection of trisomies

Question 32

What is Spectral Karyotyping (SKY)? What does SKY detect? Give an example of a disorder that can be identified with SKY?

Answer 32

• Technique “paints chromosomes” different colors with flourescence
• Detects rearrangements i.e. chromosomes that are different colors

E.g. CML i.e. 9:22 rearrangement

Question 33

What is Array-Based Comparative Genomic Hybridization (Array CGH)?

Answer 33

1) Genomic DNA & reference DNA are labeled with two different flourescent dyes
2) Hydridized to slide spotted w/ DNA probes

E.g. red & green dyes–> yellow color on hybridization indicating normal amount of both DNA

**This is a technique to detect DNA deletion or amplification i.e. under or overabundance of red or green

Question 34

What is Spectral Karyotyping (SKY)? What does SKY detect? Give an example of a disorder that can be identified with SKY?

Answer 34

• Technique “paints chromosomes” different colors with flourescence
• Detects rearrangements i.e. chromosomes that are different colors

CML i.e. 9:22 rearrangement

Question 35

What are epigenetic alterations?

Answer 35

Heritable chemical modifications of DNA or chromatin that DO NOT change the DNA sequence

*Methylation of DNA or acetylation of histones

Question 36

What does DNA methylation result in?

Answer 36

Decreased expression of gene

**Occurs at CpG islands of promoters

Question 37

What is histone deacetylation associated with?

Answer 37

Decreased gene expression

Question 38

Give three examples of diseases caused by epigenetic changes.

Answer 38

1) Fragile X-Syndrome
2) Prader-Willi
3) Angelman