Table 1.

Summary of the specifications made to Pathogenic ACMG-AMP criteria codes for the classification of variants in GAA (Version 2).

ACMG/AMP Criterion	ACMG/AMP criterion description	Specification	Strength	Description
PVS1	Null variant in a gene where loss of function is a known mechanism of disease	Gene-specific, strength	Very Strong	• Any nonsense, frameshift, or splice variant creating a premature stop codon before codon 916a. • In-frame deletions of an entire exon containing critical active site/substrate binding residuesa (exons 8 and 10), or for which another variant removing the exon is known to be pathogenic (exons 2 and 18).
			Strong	• In-frame loss of an exon which is part of the catalytic barrel domain and contains pathogenic/likely pathogenic non-truncating variants (exons 6 and 9). • Initiator codon variantb.
			Moderate	• Premature termination codon in the 3’ end of GAA (3’ to codon 916), not predicted to be degraded by nonsense-mediated decay. • Predicted exon-skipping due to canonical splice variant or exon deletion resulting in an in-frame deletion of <10% of the gene product (exons 17, 19, and 20).
PS1	Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.	N/A	Strong	No changes
PS2	De novo (maternity and paternity confirmed) in a patient with the disease and no family history.	Not used	N/A	N/A
PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect.	Gene-specific, strength	Strong	RT-PCR evidence of mis-splicing for non-canonical intronic variants with no evidence of normal splice products.
			Moderate	• <5% wild type GAA activity when the variant is expressed in a heterologous cell type and evidence of abnormal GAA synthesis and/or processing. • RT-PCR evidence of mis-splicing for non-canonical intronic variants with evidence of normal splice products.
			Supporting	• <30% wild type GAA activity when the variant is expressed in a heterologous cell type without additional evidence of abnormal synthesis and/or processing. • RT-PCR evidence of mis-splicing for non-canonical intronic variants with evidence of normal splice products.
PS4	The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.	Not used	N/A	N/A
PM1	Located in a mutational hot spot and/or critical and well-established functional domain without benign variation	Gene-specific	Moderate	Missense substitution or in frame deletion of residues important in the active site architecture and substrate binding of GAA:- Asp282, Trp376, Asp404, Leu405, Ile441, Trp481, Trp516, Asp518, Met519, Arg600, Trp613, Asp616, Trp618, Phe649, Leu650, His674.
PM2	Low frequency in population databases.		Supporting	Minor allele frequency <0.1% (0.001) in all continental populations with >2000 alleles in gnomAD.
PM3	Detected in trans with a pathogenic variant	Strength	Very Strong	4 or more points (see SVI recommendations)
			Strong	>2-4 points (see SVI recommendations)
			Moderate	2-<4 points (see SVI recommendations)
			Supporting	1-<2 points (see SVI recommendations)
PM4	Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants	Strength	Moderate	In-frame deletion/insertions of two or more amino acids but less than one exon.
			Supporting	In-frame deletion/insertions of one amino acid.
PM5	Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.	Strength	Moderate	No changes
			Supporting	Missense change at an amino acid residue where a different missense change determined to be pathogenic by the LD VCEP has been seen before.
PM6	Confirmed de novo without confirmation of paternity and maternity.	Not used	N/A	N/A
PP1	Co-segregation with disease in multiple affected family members.	Not used	N/A	N/A
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.	Not used	N/A	N/A
PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene product.	Gene-specific	Supporting	• REVEL score >0.7 for missense variants. • In-frame deletion or insertion predicted to be deleterious by 2 out of 3 tools (e.g. PROVEAN, MutationTaster, MutPred-InDel). • Predicted impact on splicing by SpliceAI (score >0.5).
PP4	Phenotype specific for disease with single genetic etiology.	Gene-specific, strength	Moderate Supporting	Points-based system Points-based system
PP5	Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation	Not used	N/A	N/A60

^a50 base pairs upstream of the penultimate exon/intron boundary, the point beyond which nonsense-mediated decay is not predicted for a premature termination codon.