Molecular pathology

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.

Contents

Utility of molecular pathology

Its utility currently includes:

  1. Proving clonality, esp. in hematologic malignancies, to help establish a malignant diagnosis.
  2. Finding recurrent genetic changes - which may be diagnostic, prognostic and suggest a specific therapy.
  3. 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

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.
  • 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).

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.[5]
    • DNA is extracted using phenol and isopropanol.[6]

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

References

  1. 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.
  2. 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.
  3. 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.
  4. URL: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml. Accessed on: 10 May 2011.
  5. 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.
  6. 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.
  7. Worldwide Protein Data Bank. URL: http://www.wwpdb.org/faq.html Accessed on: April 22, 2009.
  8. 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.
  9. 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.

External links