Table 4.

ACMG/AMP codes recommended for recording evidence relevant to variant position, and predicted and experimentally observed impact on splicing

ACMG/AMP Code	Original definition (Richards et al 2015)	(Re)definition for RNA impact	Notes
PVS1	PVS1. Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function is a known mechanism of disease.	Bioinformatic data only - Canonical splice site variants (±1,2) in a gene with established LOF as a disease mechanism.	• Develop gene-specific decision tree where feasible. • Use PVS1 decision tree to determine code strength.
PVS1_Strength (RNA)	-	Splicing assay data - Assays demonstrating a variant leads to aberrant splicing profile that can be categorised against a PVS1 decision tree.	• PVS1_Strength (RNA) to be used to designate capture of splicing data (not PS3). • Use PVS1 decision tree to determine code strength. See Figure 4 flowchart. PVS1_Strength (RNA) may not be applicable for variants for which there is a plausible rescue model
PS1	PS1. Same amino acid change as a previously established Pathogenic variant regardless of nucleotide change.	Same predicted splicing impact as a previously classified (Likely) Pathogenic variant.	• Predicted event of variant matches that of a known (Likely) Pathogenic variant. • Weights depend on relative positions of the variant under assessment and confidence in classification for the comparison variant. See Table 3.
PS3	PS3. Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.	Not applicable for splicing effects.	• Replaced by PVS1_Strength (RNA), with weight determined as per PVS1 decision tree and other factors. See Figure 4 flowchart.
PP3	PP3. Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: 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. PP3 can be used only once in any evaluation of a variant.	Computational evidence from calibrated prediction tool/s supports impact on splicing.	• No requirement for multiple tools, but calibration of tool/s to select cutoffs for predicting spliceogenicity is recommended (gene-specific calibration not required). • Only use for non-canonical splice site variants (outside of +/−1,2) when splicing assay data is not available. • For exonic variants, also consider functional impact via encoded change in protein sequence.
BS3	BS3. Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.	Not applicable for splicing effects.	• Replaced by BP7_Strong (RNA). See Figure 4 flowchart.
BP4	BP4. Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.) Caveat: Because many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.	Computational evidence from calibrated prediction tool/s supports no impact on splicing	• No requirement for multiple tools, but calibration of tool/s to select cutoffs for predicting spliceogenicity is recommended (gene-specific calibration not required). • May be applied in conjunction with BP7 and its derivatives for intronic/silent variants. See Figure 4 flowchart. • For exonic variants, also consider functional impact via encoded change in protein sequence.
BP7	BP7. A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.	Synonymous (silent) variant OR intronic variant with low prior probability of pathogenicity if no predicted impact on splicing.	• Apply only if BP4 is met. • Evolutionary conservation is not considered informative for application of this code. • Only applicable for intronic/non-coding variants outside the designated splice region (conservatively at or beyond positions +7/−21 and synonymous (silent) exonic variants located outside of the first and the last 3 bases of the exon, otherwise minimal splice region).
BP7_Strong (RNA)	-	Splicing assay data demonstrating a variant is not associated with aberrantly spliced transcript/s relative to transcript profiles in controls.	• BP7_Strong (RNA) to be used to designate capture of splicing data (not BS3). • See Figure 4 flowchart for guidance on weighting and combining with other codes. • For exonic variants, also consider functional impact via encoded change in protein sequence.