Interpreting Sequence Variants of Unknown Significance Flashcards
Variants should be described with respect to a HGVS nomenclature and what else?
With respect to a reference DNA sequence specified by an accession number and a version (e.g. NM_000059.3)
Describe some common types of pathogenic mutation
- deletions (entire gene/part of gene/single or multiple exons)
- disrupt gene structure (translocation/inversion/insertion)
- prevent promoter working (methylation)
- prevent correct splicing (inactivation of donor or acceptor splice site, activation of cryptic splice site)
- Frameshift
- produce a stop codon
- replace or delete an essential amino acid
What type of variants tend to be those of unknown clinical significance?
- Missense changes: aa changes of unknown effect
- intronic variants: not donor/acceptor site
- synonymous (silent) changes: variant does not change aa but may produce a cryptic splice site or disrupt exon splice enhancer/silencer sequences
List ten lines of evidence for investigating variants of unknown clinical significance
- Mutation databases
- Presence/absence in SNP databases
- Testing matched controls
- Literature search
- Co-occurrence in trans with a known pathogenic mutation
- Co-segregation with the disease in a family
- Loss of heterozygosity in tumour (loss of WT allele)
- Occurrence of new variant with sporadic incidence of the disease
- Species conservation
- In silico prediction software programs
What is the most commonly used mutation database when investigating unknown sequence changes?
Diagnostic Mutation Database (DMuDB) - contains variants and known mutations for many genes
What is a commonly used locus-specific mutation database for investigating unknown sequence changes?
RettBASE
What are the issues associated with using cosegregation with disease in the family for investigating unknown sequence changes?
- limited by partial Penetrance
- disorder may be late onset
- requires samples from enough family members
- is the variant linked to an unidentified pathogenic variant on the same allele?
What is the principle behind species conservation in the context of investigating unknown sequence changes?
The more conserved an amino acid is the less likely an amino acid change will be tolerated. It is recommended to include at least five mammalian homologues over an appropriate amount of evolutionary time (e.g. 300 million years)
Describe loss of heterozygosity in tumours in the context of investigating unknown sequence changes
Loss of the variant allele suggests that the unclassified variant is not pathogenic, whereas loss of the wild type allele particularly or no loss at all is consistent with pathogenicity
What does in silico prediction of pathogenic effect do in the context of unknown sequence changes? Give three examples of software used for this
This is software to predict if an amino acid substitution is tolerated
- Align GVGD
- SIFT (Sorting Intolerant From Tolerant)
- Polyphen
Describe how the in silico prediction tool align GVGD works
- combines protein sequence alignments of multiple species with biophysical characteristics of amino acids
- The biochemical distance of the variant amino acid from the observed amino acid at a particular location in different species is calculated (Grantham deviation)
- A grade, varying from C0 to C65 is given to estimate the probability that a certain variant is pathogenic (high risk >60, low risk
Describe how the in silico prediction tool SIFT works
- based on sequence evolutionary conservation of the affected residue and the type of amino acid substitution
- scores 0.05 are predicted to be tolerant (benign)
Give an example of software used for in silico splice site prediction
AlaMut: incorporates multiple in silico prediction software programs
What mutation nomenclature is used?
HGVS (Human Genome Variation Society)
