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.

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 -most labs consider fresh or frozen tissue a requirement

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):
  • 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).
  • 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.
  • DNA is extracted using phenol and isopropanol.

Other molecular tests

Techniques

• Southern blot.
  • DNA quantification.

Key elements:

• Gel electrophoresis.
• Labeling with hybridization probe.

Cytogenetics

Main article: 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. Ironically, its announcement is in a pay-for-access journal (Nature Structual Biology).

Wnt/beta-catenin pathway

Important in hepatoblastomas.

See also

• Basics.
• Chromosomal translocations.
• DNA sequence comparison.
• Tumour suppressor gene.
• Next generation sequencing.

External links

• UCSC Genome Browser (ucsc.edu).