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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. | |||
==Overview== | ==Overview== | ||
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{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}} | {{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}} | ||
{{familytree | |,|-|-|^|-|-|.|}} | {{familytree | |,|-|-|^|-|-|.|}} | ||
{{familytree | B11 | | | | B12 |B11= | {{familytree | B11 | | | | B12 |B11=Electrophoresis<br>based techniques|B12=[[Cytogenetics]]}} | ||
{{familytree/end}} | {{familytree/end}} | ||
</center> | </center> | ||
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|- | |- | ||
| in situ hybridization (ISH) | | in situ hybridization (ISH) | ||
| good way to find gene losses and duplications (one colour) and gene splits and fusions (two colours); | | '''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 | | '''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 | | karyotyping | ||
| finds large scale gains, losses | | 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) | | '''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 | | PCR + sequencing ''or'' enzyme digestion and electrophoresis | ||
| high resolution (can find very small changes, e.g. base pair substitutions); can be done on [[formalin]] fixed paraffin embedded tissue | | '''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 | | 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 techniques==== | ====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) | ||
| | | $$$ | ||
| gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced | | gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced | ||
|- | |- | ||
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| any change in the mRNA (post-splicing); useful for infering protein level changes | | any change in the mRNA (post-splicing); useful for infering protein level changes | ||
| $$$ | | $$$ | ||
| less costly than DNA sequencing - as extrons are not sequenced | | slightly less costly than DNA sequencing - as the extrons are not sequenced | ||
|- | |- | ||
| Restriction fragment length polymorphism (RFLP) | | Restriction fragment length polymorphism (RFLP) | ||
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| $$ | | $$ | ||
| value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene | | value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene | ||
|- | |- | ||
| Amplification-refractory mutation system (ARMS) | | Amplification-refractory mutation system (ARMS) | ||
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| $$ | | $$ | ||
| primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product | | primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product | ||
|- | |||
| Southern blot | |||
| gel electrophoresis, hybridization probe with label | |||
| useful for finding a specific known change, quantifying gene copy number | |||
| $$$$$ | |||
| -rarely done<br>-does '''not''' use PCR<br>-considered the gold standard for clonality<ref name=pmid10583924>{{Cite journal | last1 = Medeiros | first1 = LJ. | last2 = Carr | first2 = J. | title = Overview of the role of molecular methods in the diagnosis of malignant lymphomas. | journal = Arch Pathol Lab Med | volume = 123 | issue = 12 | pages = 1189-207 | month = Dec | year = 1999 | doi = 10.1043/0003-9985(1999)1231189:OOTROM2.0.CO;2 | PMID = 10583924 }}</ref><br>-most labs consider fresh or frozen tissue a requirement<ref name=pmid11070157>{{Cite journal | last1 = Reinartz | first1 = JJ. | last2 = McCormick | first2 = SR. | last3 = Ikier | first3 = DM. | last4 = Mellgen | first4 = AM. | last5 = Bonham | first5 = SC. | last6 = Strickler | first6 = JG. | last7 = Mendiola | first7 = JR. | title = Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue. | journal = Mol Diagn | volume = 5 | issue = 3 | pages = 227-33 | month = Sep | year = 2000 | doi = 10.1054/modi.2000.19808 | PMID = 11070157 }}</ref> | |||
|} | |} | ||
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! Other | ! Other | ||
|- | |- | ||
| ISH break apart probe (two colours) | | Interphase ISH break apart probe (two colours) | ||
| probes label two parts of a (normal) gene; the two markers straddle (common) break points | | probes label two parts of a (normal) gene; the two markers straddle (common) break points | ||
| gene fragmentation consistent with translocation; one may find: | | gene fragmentation consistent with translocation; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | ||
| $$$ | | $$$$ | ||
| can detect translocations - without knowing the specific fusion product | | can detect translocations - without knowing the specific fusion product | ||
|- | |- | ||
| ISH fusion probe (two colours) | | Interphase ISH fusion probe (two colours) | ||
| probes label different genes (that are not adjacent) | | probes label different genes (that are not adjacent) | ||
| translocation involving the two genes labeled; one may find: gene duplication, loss | | translocation involving the two genes labeled; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | ||
| $$$ | | $$$$ | ||
| can detect one specific translocation | | can detect one specific translocation | ||
|- | |- | ||
| ISH probe (one colour) | | Interphase ISH probe (one colour) | ||
| probe | | probe labels one region (gene) | ||
| gene duplication, loss | | gene duplication (or chromosomal duplication), gene loss (or chromosome loss) | ||
| $$ | | $$$ | ||
| | | | ||
|- | |- | ||
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| metaphase nuclei | | metaphase nuclei | ||
| large scale changes (fusions, deletions, translocations) | | large scale changes (fusions, deletions, translocations) | ||
| $$$ | | $$$$ | ||
| gives the "big picture" view of all the (nuclear) DNA | | gives the "big picture" view of all the (nuclear) DNA | ||
|- | |||
| Metaphase ISH probe (one colour / two colours) | |||
| probe labels one region (one colour) ''or'' probes label two parts of a (normal) gene (two colours) ''or'' probes label different genes (two colours) | |||
| gene duplication, gene loss, translocations | |||
| $$$$$ | |||
| 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=== | ===General=== | ||
*A molecular technique to duplicate DNA (or RNA) molecules ("amplify") and allow the DNA (or RNA) sequence to be determined. | |||
* | |||
**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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==Miscellaneous stuff== | ==Miscellaneous stuff== | ||
===World protein databank=== | ===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=== | ===Wnt/beta-catenin pathway=== | ||
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*[[Chromosomal translocations]]. | *[[Chromosomal translocations]]. | ||
*[[DNA sequence comparison]]. | *[[DNA sequence comparison]]. | ||
*[[Tumour suppressor gene]]. | |||
*[[Next generation sequencing]]. | |||
==References== | ==References== |
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