Exam #3: Molecular Pathology Flashcards
What are four basic applications of molecular diagnosis?
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
What is the definition of cytogenetic analysis?
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
What is cytogenetic analysis useful for detecting?
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
What is molecular analysis?
Analysis of DNA & RNA
What is molecular analysis used for?
Looking for subtle changes that cannot be detected by cytogenetics
What types of mutations is cytogenetic analysis used for?
1) Genome mutations= loss or gain of an entire chromosome
2) Chromosome mutations= rearrangement of genetic material
What type of mutations is molecular analysis used for?
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
What are the four different types of gene mutations
1) Point mutations
2) Insertions
3) Deletions
4) Trinucleotide repeat mutations
What is a trinucleotide repeat mutation?
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
List examples of diseases that are caused by trinucleotide repeat expansions.
- Myoclonus Epilepsy
- Fragile X Syndrome
- Friedreich Ataxia
- Huntington Disease
- Myotonic Dystrophy
Where are the expansions for: Myoclonus Epilepsy, Fragile X Syndrome, Friedreich Ataxia, Huntington Disease, & Myotonic Dystrophy located in the gene?
- Myoclonus Epilepsy= Promoter
- Fragile X Syndrome= UTR
- Friedreich Ataxia= Intron
- Huntington Disease= Exon
- Myotonic Dystrophy= UTR
What is pre-mRNA?
mRNA that has not be processed i.e. not spliced
What causes errors in mRNA splicing?
Single point mutations in introns can cause splicing errors
What is an example of a disease caused by a splice error of pre-mRNA?
Beta-thallassemia
After splicing, how else is pre-mRNA processed?
1) 5’ cap
2) Poly-A tail
What happens when there are mutations in the polyadenylation signal of mRNA processing?
Results in unstable mRNA
Give an example of a disease caused by a mutation in polyadenylation signal.
Beta-thallassemia
I.e. B-tallasemia can be caused by a variety of mRNA processing mutations
What is the difference between direct detection of mutations in DNA vs. indirect detection?
Direct
- Comparison of mutated sequence with known normal sequence
Indirect
- Sequence unknown
- Uses linkage markers
Outline the process of direct detection of DNA mutations.
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
Give an example of a disease that is detected with direct analysis.
Coagulation Factor V
What is allele-specific extension?
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
Outline the process of allele-specific extension/ SNP genotyping.
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
Generally, how is indirect gene analysis performed?
Linkage of the disease gene with a marker that is nearby
What does indirect linkage analysis require? What does it not require?
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
Outline the process of linkage analysis/ indirect detection.
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
What is the mnemonic to remember what southern, northern, & western blots are analyzing?
SNoW
DRoP
Southern= DNA Northern= RNA Western= Protein
What are the major three infectious agents that are detected with molecular analysis?
1) HIV
2) Hep B
3) Hep C
How is the quantification of RNA used clinically?
Guide for therapy e.g. quantification of HIV RNA is an important guide for therapy
How is molecular analysis used in forensic medicine?
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
What is FISH?
Flourescence In Situ Hybridization
How can FISH be used clincally?
Detection of trisomies
What is Spectral Karyotyping (SKY)? What does SKY detect? Give an example of a disorder that can be identified with SKY?
- Technique “paints chromosomes” different colors with flourescence
- Detects rearrangements i.e. chromosomes that are different colors
E.g. CML i.e. 9:22 rearrangement
What is Array-Based Comparative Genomic Hybridization (Array CGH)?
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
What is Spectral Karyotyping (SKY)? What does SKY detect? Give an example of a disorder that can be identified with SKY?
- Technique “paints chromosomes” different colors with flourescence
- Detects rearrangements i.e. chromosomes that are different colors
CML i.e. 9:22 rearrangement
What are epigenetic alterations?
Heritable chemical modifications of DNA or chromatin that DO NOT change the DNA sequence
*Methylation of DNA or acetylation of histones
What does DNA methylation result in?
Decreased expression of gene
**Occurs at CpG islands of promoters
What is histone deacetylation associated with?
Decreased gene expression
Give three examples of diseases caused by epigenetic changes.
1) Fragile X-Syndrome
2) Prader-Willi
3) Angelman
