Difference between revisions of "Molecular pathology"
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'''Molecular pathology''' is the | [[Image:G-Storm thermal cycler.jpg|thumb|300px|right|A thermal cycler used for PCR-based molecular testing. ([[WC]])]] | ||
'''Molecular pathology''' is the study of disease at the molecular level. It is becoming increasingly important in pathology. | |||
== | ==Utility of molecular pathology== | ||
Its utility currently includes: | |||
# Proving clonality, esp. in hematologic malignancies, to help establish a malignant diagnosis. | |||
# Finding recurrent genetic changes - which may be diagnostic, prognostic and suggest a specific therapy. | |||
# Monitor minimal residual disease. | |||
==Wnt/beta-catenin pathway== | ==Overview== | ||
Important in hepatoblastomas.<ref>PBoD | Molecular pathology can be divided as follows: | ||
<center> | |||
{{familytree/start}} | |||
{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}} | |||
{{familytree | |,|-|-|^|-|-|.|}} | |||
{{familytree | B11 | | | | B12 |B11=Electrophoresis<br>based techniques|B12=[[Cytogenetics]]}} | |||
{{familytree/end}} | |||
</center> | |||
===Tabular comparisons=== | |||
====Overview==== | |||
A simplified overview of molecular pathology: | |||
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto" | |||
! Name of technique | |||
! Advantages | |||
! Disadvantages | |||
|- | |||
| in situ hybridization (ISH) | |||
| '''intermediate resolution''' - better resolution than karyotyping for the specific target of the given ISH; good way to find gene losses and duplications (one colour) and gene splits and fusions (two colours); can be done on [[formalin]] fixed paraffin embedded tissue | |||
| '''target specific''' (if the target is wrong no information is gained ''or'' one is mislead by the negative result); NOT good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong | |||
|- | |||
| karyotyping | |||
| finds '''large scale changes''' (gains, losses, rearrangements); good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong | |||
| '''low resolution''' (completely misses small scale changes); '''requires fresh tissue/cell culture''' (as it is based on metaphase nuclei) | |||
|- | |||
| PCR + sequencing ''or'' enzyme digestion and electrophoresis | |||
| '''high resolution''' (can find very small changes, e.g. base pair substitutions) - considered gold standard; can be done on [[formalin]] fixed paraffin embedded tissue | |||
| expensive; thus, limited to small regions ('''target specific'''); enzyme digestion and electrophoresis is a compromise of sorts where one needs to know something about the expected abnormality; (gene) duplications may be difficult to prove; regions with many repeats may be difficult to sequence | |||
|} | |||
====PCR-based/electrophoresis based techniques==== | |||
A comparison of common molecular techniques: | |||
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto" | |||
! Name of technique | |||
! Key elements | |||
! Type of change detected | |||
! Cost | |||
! Other | |||
|- | |||
| DNA sequencing | |||
| PCR, sequencing machine | |||
| any (small) DNA change in the genome; does not account for post-transcriptional changes (one cannot definitively infer protein level change) | |||
| $$$ | |||
| gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced | |||
|- | |||
| RNA sequencing | |||
| reverse transcription PCR, sequencing maching | |||
| any change in the mRNA (post-splicing); useful for infering protein level changes | |||
| $$$ | |||
| slightly less costly than DNA sequencing - as the extrons are not sequenced | |||
|- | |||
| Restriction fragment length polymorphism (RFLP) | |||
| PCR, restriction endonuclease digestion, gel electrophoresis | |||
| useful for finding common base pair changes | |||
| $$ | |||
| value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene | |||
|- | |||
| Amplification-refractory mutation system (ARMS) | |||
| PCR with mutation-specific primer, gel electrophoresis | |||
| useful for finding a specific known change | |||
| $$ | |||
| primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product | |||
|- | |||
| Southern blot | |||
| gel electrophoresis, hybridization probe with label | |||
| useful for finding a specific known change, quantifying gene copy number | |||
| $$$$$ | |||
| -rarely done<br>-does '''not''' use PCR<br>-considered the gold standard for clonality<ref name=pmid10583924>{{Cite journal | last1 = Medeiros | first1 = LJ. | last2 = Carr | first2 = J. | title = Overview of the role of molecular methods in the diagnosis of malignant lymphomas. | journal = Arch Pathol Lab Med | volume = 123 | issue = 12 | pages = 1189-207 | month = Dec | year = 1999 | doi = 10.1043/0003-9985(1999)1231189:OOTROM2.0.CO;2 | PMID = 10583924 }}</ref><br>-most labs consider fresh or frozen tissue a requirement<ref name=pmid11070157>{{Cite journal | last1 = Reinartz | first1 = JJ. | last2 = McCormick | first2 = SR. | last3 = Ikier | first3 = DM. | last4 = Mellgen | first4 = AM. | last5 = Bonham | first5 = SC. | last6 = Strickler | first6 = JG. | last7 = Mendiola | first7 = JR. | title = Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue. | journal = Mol Diagn | volume = 5 | issue = 3 | pages = 227-33 | month = Sep | year = 2000 | doi = 10.1054/modi.2000.19808 | PMID = 11070157 }}</ref> | |||
|} | |||
====Cytogenetics==== | |||
A comparison of ISH and karyotyping: | |||
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto" | |||
! Name of technique | |||
! Key elements | |||
! Type of change detected | |||
! Cost | |||
! Other | |||
|- | |||
| Interphase ISH break apart probe (two colours) | |||
| probes label two parts of a (normal) gene; the two markers straddle (common) break points | |||
| gene fragmentation consistent with translocation; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | |||
| $$$$ | |||
| can detect translocations - without knowing the specific fusion product | |||
|- | |||
| Interphase ISH fusion probe (two colours) | |||
| probes label different genes (that are not adjacent) | |||
| translocation involving the two genes labeled; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | |||
| $$$$ | |||
| can detect one specific translocation | |||
|- | |||
| Interphase ISH probe (one colour) | |||
| probe labels one region (gene) | |||
| gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | |||
| $$$ | |||
| | |||
|- | |||
| Karyotyping | |||
| metaphase nuclei | |||
| large scale changes (fusions, deletions, translocations) | |||
| $$$$ | |||
| gives the "big picture" view of all the (nuclear) DNA | |||
|- | |||
| Metaphase ISH probe (one colour / two colours) | |||
| probe labels one region (one colour) ''or'' probes label two parts of a (normal) gene (two colours) ''or'' probes label different genes (two colours) | |||
| gene duplication, gene loss, translocations | |||
| $$$$$ | |||
| rarely done; follows karyotyping to better characterize unusual cases; can be thought of as a karyotype and a simultaneous ISH | |||
|} | |||
==Data formats== | |||
Human gene naming is provided by the HUGO Gene Nomenclature Committee: https://www.genenames.org/ | |||
DNA data repositories | |||
* NCBI: National Center for Biotechnology Information | |||
**Standard sequencing data is usually located in Nucleotide database: https://www.ncbi.nlm.nih.gov/nuccore | |||
**Next-gen sequencing data is in short read archive: https://www.ncbi.nlm.nih.gov/sra/docs/submit/ | |||
* EMBL: European Molecular Biology Laboratory | |||
* DDBJ: DNA Data Bank of Japan | |||
DNA sequence data formats | |||
* GenBank: human readable, can be processed by computer (fixed width, first 10 characters are an identifier). | |||
** NCBI Reference Sequence (RefSeq) project provides sequence records and related information. | |||
** Prefix AC_ in acession number is for genomic data, NM_ is for mRNA. | |||
* FASTA: Sequence information | |||
** Header starts with > and is followed by a sequence ID. | |||
** Sequence lines should wrap always at the same width. | |||
** Lower-case letters may indicate repetitive regions. | |||
* FASTQ: Current standard for sequencing data | |||
** It is essentially FASTA with quality values for the sequence. | |||
** Quality is on a scale from 0 - 40 and represented by a distinct character. | |||
** Upper case letters ABCDEFGHI means high quality. | |||
** Special letters !"#$%&'()*+,-. mean low quality. | |||
==Polymerase chain reaction-based techniques== | |||
:Abbreviated ''PCR-based techniques'' | |||
:''PCR'' redirects here | |||
===General=== | |||
*A molecular technique to duplicate DNA (or RNA) molecules ("amplify") and allow the DNA (or RNA) sequence to be determined. | |||
Utility? | |||
*Detect very small molecular changes - submicroscopic. | |||
**Changes in sequence - may be as small as one base pair. | |||
*Used to confirmation [[chromosomal translocation]]s that are, in clinical practice, usually found with other techniques. | |||
===Techniques=== | |||
*DNA sequencing. | |||
**Real time-PCR, [[AKA]] real time-quantitative PCR (RQ-PCR). | |||
*RNA sequencing. | |||
**May be examined after reverse transcription (RNA -> DNA), i.e. RT-PCR. | |||
*Amplification-refractory mutation system (ARMS):<ref name=pmid18428319>{{cite journal |author=Little S |title=Amplification-refractory mutation system (ARMS) analysis of point mutations |journal=Curr Protoc Hum Genet |volume=Chapter 9 |issue= |pages=Unit 9.8 |year=2001 |month=May |pmid=18428319 |doi=10.1002/0471142905.hg0908s07 |url=}}</ref> | |||
**Technique for finding a (specific) single base change. | |||
***The (PCR) primers are designed bind to the mutated sequence. | |||
****If the mutation is present a PCR product is seen. | |||
****If the mutation is absent no PCR product is seen. | |||
*Restriction fragment length polymorphism (RFLP).<ref>URL: [http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml]. Accessed on: 10 May 2011.</ref> | |||
**Technique useful for finding a single base change. | |||
***Restriction endonuclease(s), generally, will generate different fragment lengths if nucleotide change is present. | |||
***This techique is most useful if one is looking for a specific (small) genetic change (e.g. F5 Arg534Gln). | |||
====Specific tests==== | |||
A list of tests are found in the ''[[Molecular pathology tests]]'' article. | |||
====DNA & RNA extraction==== | |||
*Techniques are largely standardized. | |||
*Protocols exist for fresh tissue and [[formalin]] fixed paraffin imbedded tissue. | |||
**RNA is usually extracted with acid guanidium thiocyanate, phenol and choroform.<ref>{{cite journal |author=Chomczynski P, Sacchi N |title=The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on |journal=Nat Protoc |volume=1 |issue=2 |pages=581–5 |year=2006 |pmid=17406285 |doi=10.1038/nprot.2006.83 |url=}}</ref> | |||
**DNA is extracted using phenol and isopropanol.<ref>{{cite journal |author=Pikor LA, Enfield KS, Cameron H, Lam WL |title=DNA extraction from paraffin embedded material for genetic and epigenetic analyses |journal=J Vis Exp |volume= |issue=49 |pages= |year=2011 |pmid=21490570 |doi=10.3791/2763 |url=}}</ref> | |||
==Other molecular tests== | |||
===Techniques=== | |||
*Southern blot. | |||
**DNA quantification. | |||
Key elements: | |||
*Gel electrophoresis. | |||
*Labeling with hybridization probe. | |||
==Cytogenetics== | |||
{{Main|Cytogenetics}} | |||
This deals with karyotyping and ISH. | |||
==Miscellaneous stuff== | |||
===World protein databank=== | |||
The protein databank's goal is to maintain a free and publicly available archive.<ref>Worldwide Protein Data Bank. URL: [http://www.wwpdb.org/faq.html http://www.wwpdb.org/faq.html] Accessed on: April 22, 2009.</ref> Ironically, its announcement is in a pay-for-access journal (''Nature Structual Biology'').<ref name=pmid14634627>{{cite journal |author=Berman H, Henrick K, Nakamura H |title=Announcing the worldwide Protein Data Bank |journal=Nat. Struct. Biol. |volume=10 |issue=12 |pages=980 |year=2003 |month=December |pmid=14634627 |doi=10.1038/nsb1203-980 |url=}}</ref> | |||
===Wnt/beta-catenin pathway=== | |||
Important in hepatoblastomas.<ref name=Ref_PBoD923>{{Ref PBoD|923}}</ref> | |||
==See also== | ==See also== | ||
*[[Basics]] | *[[Basics]]. | ||
*[[Chromosomal translocations]]. | |||
*[[DNA sequence comparison]]. | |||
*[[Tumour suppressor gene]]. | |||
*[[Next generation sequencing]]. | |||
==References== | ==References== | ||
{{reflist|2}} | {{reflist|2}} | ||
==External links== | |||
*[http://genome.ucsc.edu/ UCSC Genome Browser (ucsc.edu)]. | |||
[[Category:Molecular pathology]] | [[Category:Molecular pathology]] |
Latest revision as of 20:24, 11 October 2023
![](/w/images/thumb/b/b8/G-Storm_thermal_cycler.jpg/300px-G-Storm_thermal_cycler.jpg)
Molecular pathology is the study of disease at the molecular level. It is becoming increasingly important in pathology.
Utility of molecular pathology
Its utility currently includes:
- Proving clonality, esp. in hematologic malignancies, to help establish a malignant diagnosis.
- Finding recurrent genetic changes - which may be diagnostic, prognostic and suggest a specific therapy.
- Monitor minimal residual disease.
Overview
Molecular pathology can be divided as follows:
Molecular pathology | |||||||||||||||||||
Electrophoresis based techniques | Cytogenetics | ||||||||||||||||||
Tabular comparisons
Overview
A simplified overview of molecular pathology:
Name of technique | Advantages | Disadvantages |
---|---|---|
in situ hybridization (ISH) | intermediate resolution - better resolution than karyotyping for the specific target of the given ISH; good way to find gene losses and duplications (one colour) and gene splits and fusions (two colours); can be done on formalin fixed paraffin embedded tissue | target specific (if the target is wrong no information is gained or one is mislead by the negative result); NOT good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong |
karyotyping | finds large scale changes (gains, losses, rearrangements); good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong | low resolution (completely misses small scale changes); requires fresh tissue/cell culture (as it is based on metaphase nuclei) |
PCR + sequencing or enzyme digestion and electrophoresis | high resolution (can find very small changes, e.g. base pair substitutions) - considered gold standard; can be done on formalin fixed paraffin embedded tissue | expensive; thus, limited to small regions (target specific); enzyme digestion and electrophoresis is a compromise of sorts where one needs to know something about the expected abnormality; (gene) duplications may be difficult to prove; regions with many repeats may be difficult to sequence |
PCR-based/electrophoresis based techniques
A comparison of common molecular techniques:
Name of technique | Key elements | Type of change detected | Cost | Other |
---|---|---|---|---|
DNA sequencing | PCR, sequencing machine | any (small) DNA change in the genome; does not account for post-transcriptional changes (one cannot definitively infer protein level change) | $$$ | gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced |
RNA sequencing | reverse transcription PCR, sequencing maching | any change in the mRNA (post-splicing); useful for infering protein level changes | $$$ | slightly less costly than DNA sequencing - as the extrons are not sequenced |
Restriction fragment length polymorphism (RFLP) | PCR, restriction endonuclease digestion, gel electrophoresis | useful for finding common base pair changes | $$ | value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene |
Amplification-refractory mutation system (ARMS) | PCR with mutation-specific primer, gel electrophoresis | useful for finding a specific known change | $$ | primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product |
Southern blot | gel electrophoresis, hybridization probe with label | useful for finding a specific known change, quantifying gene copy number | $$$$$ | -rarely done -does not use PCR -considered the gold standard for clonality[1] -most labs consider fresh or frozen tissue a requirement[2] |
Cytogenetics
A comparison of ISH and karyotyping:
Name of technique | Key elements | Type of change detected | Cost | Other |
---|---|---|---|---|
Interphase ISH break apart probe (two colours) | probes label two parts of a (normal) gene; the two markers straddle (common) break points | gene fragmentation consistent with translocation; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | $$$$ | can detect translocations - without knowing the specific fusion product |
Interphase ISH fusion probe (two colours) | probes label different genes (that are not adjacent) | translocation involving the two genes labeled; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | $$$$ | can detect one specific translocation |
Interphase ISH probe (one colour) | probe labels one region (gene) | gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | $$$ | |
Karyotyping | metaphase nuclei | large scale changes (fusions, deletions, translocations) | $$$$ | gives the "big picture" view of all the (nuclear) DNA |
Metaphase ISH probe (one colour / two colours) | probe labels one region (one colour) or probes label two parts of a (normal) gene (two colours) or probes label different genes (two colours) | gene duplication, gene loss, translocations | $$$$$ | rarely done; follows karyotyping to better characterize unusual cases; can be thought of as a karyotype and a simultaneous ISH |
Data formats
Human gene naming is provided by the HUGO Gene Nomenclature Committee: https://www.genenames.org/
DNA data repositories
- NCBI: National Center for Biotechnology Information
- Standard sequencing data is usually located in Nucleotide database: https://www.ncbi.nlm.nih.gov/nuccore
- Next-gen sequencing data is in short read archive: https://www.ncbi.nlm.nih.gov/sra/docs/submit/
- EMBL: European Molecular Biology Laboratory
- DDBJ: DNA Data Bank of Japan
DNA sequence data formats
- GenBank: human readable, can be processed by computer (fixed width, first 10 characters are an identifier).
- NCBI Reference Sequence (RefSeq) project provides sequence records and related information.
- Prefix AC_ in acession number is for genomic data, NM_ is for mRNA.
- FASTA: Sequence information
- Header starts with > and is followed by a sequence ID.
- Sequence lines should wrap always at the same width.
- Lower-case letters may indicate repetitive regions.
- FASTQ: Current standard for sequencing data
- It is essentially FASTA with quality values for the sequence.
- Quality is on a scale from 0 - 40 and represented by a distinct character.
- Upper case letters ABCDEFGHI means high quality.
- Special letters !"#$%&'()*+,-. mean low quality.
Polymerase chain reaction-based techniques
- Abbreviated PCR-based techniques
- PCR redirects here
General
- A molecular technique to duplicate DNA (or RNA) molecules ("amplify") and allow the DNA (or RNA) sequence to be determined.
Utility?
- Detect very small molecular changes - submicroscopic.
- Changes in sequence - may be as small as one base pair.
- Used to confirmation chromosomal translocations that are, in clinical practice, usually found with other techniques.
Techniques
- DNA sequencing.
- Real time-PCR, AKA real time-quantitative PCR (RQ-PCR).
- RNA sequencing.
- May be examined after reverse transcription (RNA -> DNA), i.e. RT-PCR.
- Amplification-refractory mutation system (ARMS):[3]
- Technique for finding a (specific) single base change.
- The (PCR) primers are designed bind to the mutated sequence.
- If the mutation is present a PCR product is seen.
- If the mutation is absent no PCR product is seen.
- The (PCR) primers are designed bind to the mutated sequence.
- Technique for finding a (specific) single base change.
- Restriction fragment length polymorphism (RFLP).[4]
- Technique useful for finding a single base change.
- Restriction endonuclease(s), generally, will generate different fragment lengths if nucleotide change is present.
- This techique is most useful if one is looking for a specific (small) genetic change (e.g. F5 Arg534Gln).
- Technique useful for finding a single base change.
Specific tests
A list of tests are found in the Molecular pathology tests article.
DNA & RNA extraction
- Techniques are largely standardized.
- Protocols exist for fresh tissue and formalin fixed paraffin imbedded tissue.
Other molecular tests
Techniques
- Southern blot.
- DNA quantification.
Key elements:
- Gel electrophoresis.
- Labeling with hybridization probe.
Cytogenetics
This deals with karyotyping and ISH.
Miscellaneous stuff
World protein databank
The protein databank's goal is to maintain a free and publicly available archive.[7] Ironically, its announcement is in a pay-for-access journal (Nature Structual Biology).[8]
Wnt/beta-catenin pathway
Important in hepatoblastomas.[9]
See also
- Basics.
- Chromosomal translocations.
- DNA sequence comparison.
- Tumour suppressor gene.
- Next generation sequencing.
References
- ↑ Medeiros, LJ.; Carr, J. (Dec 1999). "Overview of the role of molecular methods in the diagnosis of malignant lymphomas.". Arch Pathol Lab Med 123 (12): 1189-207. doi:10.1043/0003-9985(1999)1231189:OOTROM2.0.CO;2. PMID 10583924.
- ↑ Reinartz, JJ.; McCormick, SR.; Ikier, DM.; Mellgen, AM.; Bonham, SC.; Strickler, JG.; Mendiola, JR. (Sep 2000). "Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue.". Mol Diagn 5 (3): 227-33. doi:10.1054/modi.2000.19808. PMID 11070157.
- ↑ Little S (May 2001). "Amplification-refractory mutation system (ARMS) analysis of point mutations". Curr Protoc Hum Genet Chapter 9: Unit 9.8. doi:10.1002/0471142905.hg0908s07. PMID 18428319.
- ↑ URL: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml. Accessed on: 10 May 2011.
- ↑ Chomczynski P, Sacchi N (2006). "The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on". Nat Protoc 1 (2): 581–5. doi:10.1038/nprot.2006.83. PMID 17406285.
- ↑ Pikor LA, Enfield KS, Cameron H, Lam WL (2011). "DNA extraction from paraffin embedded material for genetic and epigenetic analyses". J Vis Exp (49). doi:10.3791/2763. PMID 21490570.
- ↑ Worldwide Protein Data Bank. URL: http://www.wwpdb.org/faq.html Accessed on: April 22, 2009.
- ↑ Berman H, Henrick K, Nakamura H (December 2003). "Announcing the worldwide Protein Data Bank". Nat. Struct. Biol. 10 (12): 980. doi:10.1038/nsb1203-980. PMID 14634627.
- ↑ Cotran, Ramzi S.; Kumar, Vinay; Fausto, Nelson; Nelso Fausto; Robbins, Stanley L.; Abbas, Abul K. (2005). Robbins and Cotran pathologic basis of disease (7th ed.). St. Louis, Mo: Elsevier Saunders. pp. 923. ISBN 0-7216-0187-1.