The NM_000152.5:c.1003G>A variant in GAA is a missense variant predicted to cause substitution of glycine by arginine at amino acid 335 (p.Gly335Arg). At least 4 patient(s) with this variant had documented GAA deficiency with <10% of normal mean control level of GAA activity in leukocytes, lymphocytes, muscle samples or were reported to be on enzyme replacement therapy for Pompe disease (PP4_Moderate) (PMIDs 31510962, 26497565, 31193175, 24685124). One of these probands is compound heterozygous for the variant and a variant classified as pathogenic by the ClinGen LSD VCEP, c.876C>G p.Tyr292X (PMID 31510962); the variants were confirmed in trans by familial testing (PM3). The highest population minor allele frequency in gnomAD v2.1.1 for this variant is 0.00003266 (1/30616 alleles) in the South Asian population, which is lower than the ClinGen LSD VCEP’s threshold for PM2_Supporting (<0.001), meeting this criterion (PM2_Supporting). Expression of the variant in COS7 or HEK293 cells resulted in 0.8% GAA activity in cells and evidence of abnormal synthesis and processing on Western blot, indicating that this variant may impact protein function (PMID 22644586)(PS3_Moderate). The computational predictor REVEL gives a score of 0.919 which is above the threshold of 0.7, evidence that correlates with impact to GAA function (PP3). Another missense variant, c.1004G>A (p.Gly335Glu), in the same codon has been reported in a patient with Pompe disease (PMIDs 22644586, 32711049)s. However, this variant has not yet met the criteria to be classified as pathogenic or likely pathogenic by the ClinGen LSD VCEP and, therefore, this evidence is not sufficient to meet PM5 at any strength. There is a ClinVar entry for this variant (Variation ID: 972790; 1 star review status) with one submitter classifying the variant as pathogenic. In summary, this variant meets the criteria to be classified as Likely Pathogenic for Pompe disease. GAA-specific ACMG/AMP criteria met, based on the specifications of the ClinGen LSD VCEP (Specifications Version 2.0): PM3, PS3_Moderate, PP4_Moderate, PP3, PM2_Supporting. (Classification approved by the ClinGen LSD VCEP, February 7, 2023).