Standardization of Molecular Assays Flashcards
What are the typical molecular defects of interest
Gene variations- deletions, insertions, rearrangements
Single nucleotide variants
Epigenetic changes- DNA methylation changes: CpG, Protein methylation changes: histones
Prevent confusion
Do not use mutation, instead use variant or disease-associated variant ; do not use polymorphism, instead use variant or not disease-associated variant
Neutral Terms
Sequence variation alternative- copy number variant (CNV), single nucleotide variant (SNV)
Nomenclature
gene name: each unique gene symbol comprises a short representation of the descriptive name, uses only Latin letters and Arabic number, no punctuation or species reference, Ex.) DAL-1=differentially expressed in Adenocarcinoma of the lung
Nomenclature
Gene family- hierarchical series used (HOXA13; HOXB13), Gene references are italicized but protein references are not, pseudogenes used the gene name of the gene they are similar to follow by a P
Variation types
change in sequence, change in amount, change in position
Nucleotide changes
Nucleotide Substitutions
A of initiation sequence ATG is position 1
Immediately preceding base is -1
Beginning of intron c.77+1G
End of intron c.78-1G
Nucleotide number followed by base change
Example: c.162 G>A
Guanine at position 162 of the coding sequence
is replaced by adenine
Nucleotide changes
-Deletions, Insertions, Duplications
*DNA changes: Nucleotide position or interval
followed by change
*Deletion Example: c.232_234delAGC
*Del of 3 nucleic acids starting at position 232
*Insertion Example: c.232_233insG
*Insertion of 1 nucleotide after position 232
*Duplication: c.232dupA
*Duplication of an A after position 232
Amino Acid Changes
Amino acid substitutions
- Original amino acid, position, new amino acid
ex. ) p.Arg117His (p.R117H) - Arginine at position 117 is replaced by histidine
Amino acid changes
Amino Acid deletion
- Amino acid followed by position ex.) F508del or ^
- Deletion of phenylalanine at position 508
Web based resources
HUGO gene nomenclature committee - HGNC
Human genome variation society -HGVS
GTR data
Includes info from resources s.a. ClinVa and Med gen from within the NIH and many resources from outside the NIH
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What does lab practice require?
Full characterization of an assay before it is used for patient testing, irrespective of federally-designated test complexity and without regard to when it was first introduced by a given lab.
The lab should have what type of data?
Test analytic sensitivity, precision and interferences as applicable, the data can originate from manufacturers’ information, published reports and/or the labs own studies
Verification and standardization
- Most methods use PCR amplification followed by electrophoretic or chromatographaphic separation
- FDA typically approves insturmentation for clinical labs; approval of molecular diagnostic kits are rare due to lack of universal standards (LDTs or homebrew)
