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Molecular pathology (view source)

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'''Molecular pathology''' is the future of pathology.
[[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.  


==Overview==
==Overview==
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{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}}
{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}}
{{familytree | |,|-|-|^|-|-|.|}}
{{familytree | |,|-|-|^|-|-|.|}}
{{familytree | B11 | | | | B12 |B11=PCR-based<br>techniques|B12=[[Cytogenetics]]}}
{{familytree | B11 | | | | B12 |B11=Electrophoresis<br>based techniques|B12=[[Cytogenetics]]}}
{{familytree/end}}
{{familytree/end}}
</center>
</center>


===Tabular comparisons===
===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:
A comparison of common molecular techniques:
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
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| PCR, sequencing machine
| PCR, sequencing machine
| any (small) DNA change in the genome; does not account for post-transcriptional changes (one cannot definitively infer protein level change)
| 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
| gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced
|-
|-
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| any change in the mRNA (post-splicing); useful for infering protein level changes
| any change in the mRNA (post-splicing); useful for infering protein level changes
| $$$
| $$$
| less costly than DNA sequencing - as extrons are not sequenced
| slightly less costly than DNA sequencing - as the extrons are not sequenced
|-
|-
| Restriction fragment length polymorphism (RFLP)
| Restriction fragment length polymorphism (RFLP)
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| $$
| $$
| value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene
| value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene
|-
| Southern blot
| gel electrophoresis, hybridization probe with label
| useful for finding a specific known change, quantifying gene copy number
| $$$
|
|-
|-
| Amplification-refractory mutation system (ARMS)
| Amplification-refractory mutation system (ARMS)
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| $$
| $$
| primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product
| primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product
|-
| Southern blot
| gel electrophoresis, hybridization probe with label
| useful for finding a specific known change, quantifying gene copy number
| $$$$$
| -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:
A comparison of ISH and karyotyping:
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
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! Other
! Other
|-
|-
| ISH break apart probe
| Interphase ISH break apart probe (two colours)
| probes label two parts of a (normal) gene; the two markers straddle (common) break points  
| probes label two parts of a (normal) gene; the two markers straddle (common) break points  
| gene fragmentation consistent with translocation; one may find: loss of part of the gene, gene duplication
| gene fragmentation consistent with translocation; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss)
| $$$
| $$$$
| can detect translocations - without knowing the specific fusion product
| can detect translocations - without knowing the specific fusion product
|-
|-
| ISH fusion probe
| Interphase ISH fusion probe (two colours)
| probes label different genes (that are not adjacent)
| probes label different genes (that are not adjacent)
| translocation involving the two genes labeled; one may find: gene duplication, loss of a gene)
| translocation involving the two genes labeled; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss)
| $$$
| $$$$
| can detect one specific translocation
| can detect one specific translocation
|-
|-
| ISH one colour
| Interphase ISH probe (one colour)
| probe label one region (gene)  
| probe labels one region (gene)  
| gene duplication, loss of a gene
| gene duplication (or chromosomal duplication), gene loss (or chromosome loss)
| $$$
| $$$
|  
|  
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| metaphase nuclei  
| metaphase nuclei  
| large scale changes (fusions, deletions, translocations)
| large scale changes (fusions, deletions, translocations)
| $$$
| $$$$
| gives the "big picture" view of the gene
| gives the "big picture" view of all the (nuclear) DNA
|-
| Metaphase ISH probe (one colour / two colours)
| probe labels one region (one colour) ''or'' probes label two parts of a (normal) gene (two colours) ''or'' probes label different genes (two colours)
| gene duplication, gene loss, translocations
| $$$$$
| rarely done; follows karyotyping to better characterize unusual cases; can be thought of as a karyotype and a simultaneous ISH
|}
|}


==PCR-based techniques==
 
==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===
===General===
What?
*A molecular technique to duplicate DNA (or RNA) molecules ("amplify") and allow the DNA (or RNA) sequence to be determined.
*Very small changes - submicroscopic.
 
**Changes in sequence  
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===
===Techniques===
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====DNA & RNA extraction====
====DNA & RNA extraction====
*Techniques are largely standardized.
*Techniques are largely standardized.
*Protocols exist for fresh tissue and formulin fixed parafin imbeded tissue.
*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>
**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>
**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>
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==Miscellaneous stuff==
==Miscellaneous stuff==
===World protein databank===
===World protein databank===
I can't help think it is ironic that the protein databank 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> yet the announcement is in 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>
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===
===Wnt/beta-catenin pathway===
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*[[Chromosomal translocations]].
*[[Chromosomal translocations]].
*[[DNA sequence comparison]].
*[[DNA sequence comparison]].
*[[Tumour suppressor gene]].


==References==
==References==
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