The NM_000152.5:c.546+1G>T variant in GAA occurs within the canonical splice donor site of intron 2. Two studies involving minigene analysis have showed that the variant disrupts normal splicing, resulting in skipping of exon 2 (PMID: 31301153, 39905333). Exon 2 contains the start methionine and signal sequence for GAA (amino acids 1-27; https://www.uniprot.org/uniprot/P10253). Deletion of exon 2 causes complete loss of enzyme activity when expressed in heterologous cells (Huie et al, 1994, PMID 7881425; Boerkoel et al, 1995, PMID 7717400) (PVS1). At least 5 probands with symptoms consistent with late-onset Pompe disease have been reported with this variant (PMID: 18285536, 25396301, 25712382, 28838325), one with documented GAA deficiency with <10% of normal mean control level of GAA activity in a muscle sample (PP4_Moderate). Of those individuals, 4 were compound heterozygous for this variant and another variant in GAA, c.-32-13T>G (ClinVar Variation ID: 4027), which is classified as pathogenic by the ClinGen Lysosomal Diseases VCEP. The phase is unconfirmed in all cases (PMID: 18285536, 25396301, 25712382, 28838325) (PM3). Additionally, this variant is absent in gnomAD v4.1.0. (PM2_Supporting). There is a ClinVar entry for this variant (Variation ID: 3241650). In summary, this variant meets the criteria to be classified as pathogenic for Pompe disease based on the GAA-specific ACMG/AMP criteria applied, as specified by the ClinGen Lysosomal Diseases Variant Curation Expert Panel (Specifications Version 2.0.0): PVS1, PM2_Supporting, PM3, PP4_Moderate. (Classification approved by the ClinGen Lysosomal Diseases Variant Curation Expert Panel on July 1, 2025)