CGC / ClinGen / VICC Classification Calculator + Emory Modification

• Considering ClinGen guidance on the interpretation of loss-of-function variants.
• Use caution interpreting pLOF variants at the extreme 3' end of a gene after the nonsense mediated decay site.
• Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact (in frame events).
• MutSpliceDB maybe helpful to catalog RNA-sequencing based evidence for splice site variants effects on splicing.
• Use caution if splice variant leads to expression of a well-known alternative iso-form that preserves tumor suppressor functionality.
• Use caution in the presence of multiple transcripts.

• Beware of changes that impact splicing rather than the changes at the amino acid/protein level.

• Functional studies that have been shown to be reproducible and robust are considered the most well established.
• If OS1 is applicable, this rule can be used only if functional studies are based on the particular nucleotide change of the variant.

• Use caution with hotspots driven by truncating somatic variants.
• Cancerhotspot has good coverage for many solid tumors; however, it has limited coverage for hematological, rare, and pediatric malignancies. If the somatic variant is in a tumor type not well covered by cancerhotspots.org, resources such as COSMIC or a tumor type-specific study could be used. Note that data in COSMIC is not adjusted by nucleotide mutability and varying gene-specific SNV rates.
• For pediatric malignancies resources, PeCanPIE may be considered.
• This rule cannot be used if OS1 is applicable, unless it is possible to observe hotspots on the basis of the particular nucleotide change.

• This rule cannot be used if OS1 or OS3 is applicable.

• This rule cannot be used if OVS1 is applicable

• Example: p.Arg156His is oncogenic; now you observe p.Arg156Cys. This rule cannot be used if OS1 or OS3 or OM1 is applicable.
• Beware of changes that impact splicing rather than the changes at the amino acid/protein level.

• The difference of variant AA from reference AA should be greater or at least approximately the same as for the missense change that has already been determined to be oncogenic. See Grantham's Distance or Miyata’s distance
• This rule cannot be used if OM1 or OM3 is applicable.
• Use caution with hotspots driven by truncating somatic variants.
• If the somatic variant is in a tumor type that is not covered well by cancerhotspots.org, resources such as COSMIC or a tumor type-specific study could be used.

• Because many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion.
• Can be used only once in any evaluation of a variant.

Conservation analysis

phyloP and phastCons - Reference

Splicing effect predictors

spliceAI - Reference

varSEAK

In silico variant effect prediction algorithms for missense variants

CADD (Meta-predictor) - Reference - Secondary reference

CHASMPlus - Reference

Dann Coding (no link) - Reference

FATHMM - Reference - Secondary reference

FATHMM MKL - Reference

FATHMM XF Coding - Reference

Likelihood Ratio Test (no link) - Reference

MetaLR (Meta-predictor, no link) - Reference

MetaSVM (Meta-predictor, no link) - Reference

Mutation Assessor - Reference

MutPred - Reference

PhD-SNPg - Reference

Polyphen HDIV - Reference

Polyphen HVAR - Reference

PROVEAN - Reference

REVEL (Meta-predictor) - Reference

SIFT - Reference

VEST - Reference

• A small fraction of cases may be caused by an alternative mechanism; histological similarities may cause misdiagnosis.

• Use caution with hotspots driven by truncating somatic variants.
• If somatic variant is in a tumor type that is not covered well by cancerhotspots.org, resources such as COSMIC or a tumor type–specific study could be used.

• Population data for insertions/deletions may be poorly called by next-generation sequencing. Population data may contain somatic variants associated with clonal hematopoiesis.

• In cases with low TMB, any mutation should be carefully evaluated because it is unlikely to be a random variant or VUS.

• Compound heterozygous mutations in tumor suppressor genes would more likely result in loss of function.

• Similar to OP5, somatic mutations in cases with low TMB are more likely oncogenic and should be evaluated carefully.

• If the somatic variant is in a gene known to cause predisposition to hereditary cancer, ACMG/AMP ClinGen germline expert panel gene-specific guidelines (if they exist) must be consulted to establish a cutoff that takes disease prevalence into account.

• Indels may be underrepresented in gnomAD in part due to technical challenges associated with their detection.
• Some variants in gnomAD (and similar databases) may actually be somatic and not germline, owing to clonal hematopoiesis (CHIP).
• If the somatic variant is in a gene known to cause predisposition to hereditary cancer, ACMG/AMP ClinGen germline expert panel gene-specific guidelines (if they exist) must be consulted to establish a cutoff that takes disease prevalence into account.

• Because many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. Can be used only once in any evaluation of a variant.