The NM_000152.5:c.1561G>A variant in GAA is a missense variant predicted to cause substitution of glutamic acid by lysine at amino acid 521 (p.Glu521Lys). This substitution of a negatively charged, polar amino acid with a neutral, non-polar amino acid is predicted to functionally impact the GAA precursor protein (PMID: 1898413). This variant was first reported in homozygosity in a case report of siblings with IOPD and no detectable GAA activity in fibroblasts (PMID: 1898413), meeting criteria for PP4_moderate. This variant has been detected in at least four additional individuals with IOPD. Of those individuals, two were compound heterozygous for the variant and a pathogenic or likely pathogenic variant (PMIDs: 17723315, 37542277) and two were homozygous for the variant (PMIDs: 19862843,33301762). This variant has also been reported in at least nine individuals with LOPD. Of those, six were compound heterozygous for the common IVS splice site pathogenic variant c.-32-13T>G (PMIDs: 22676651, 20308911, 2270448, 20033296, 25673129, 30312517,34852371 and 39045638), and one was compound heterozygous for the variant and a pathogenic or likely pathogenic variant (PMID:21605996 and 22676651) (PM3_Very Strong). The highest population minor allele frequency in gnomAD v4.1.0. is 0.00003334 (2/59992 alleles) in the Admixed American population, which is lower than the ClinGen Lysosomal Diseases VCEP’s threshold for PM2_Supporting (<0.001), meeting this criterion (PM2_Supporting). In the first report of this variant, transient expression in COS cells was performed and revealed no detectable activity, and immunocytochemistry results suggested the mutant precursor is not transported to the lysosome (PMID: 1898413). Subsequent expression studies in COS cells revealed <2% wild type GAA activity, indicating that this variant may impact protein function (PMID: 19862843). Thus, PS3_Supporting can be applied. The computational predictor REVEL gives a score of 0.932 which is above the threshold of 0.7, evidence that correlates with impact to GAA function (PP3). Another missense change at the same amino acid position (p.Glu521Gln) has been reported in IOPD (PMID: 22658377, 19588081), but this variant has not been reviewed by the ClinGen Lysosomal Diseases Variant Curation Expert panel and thus PM5 will not be applied. There is a ClinVar entry for this variant (Variation ID: 4022, 2-star review status) with 8 submitters classifying the variant as pathogenic (7 submitters) or likely pathogenic (1 submitters). In summary, this variant meets the criteria to be classified as pathogenic for Pompe disease based on the ACMG/AMP criteria applied, as specified by the ClinGen Lysosomal Diseases Variant Curation Expert panel (Specifications Version 2.0): PM3_Very Strong, PP4_moderate, PM2_supporting, PP3, PS3_supporting. (Classification approved by the ClinGen Lysosomal Diseases Variant Curation Expert Panel on December 3, 2024)

The p.Glu521Lys variant in GAA has been reported in 11 individuals (including at least 3 Caucasian and 2 Indian individuals) with Glycogen Storage Disease II, segregated with disease in 2 affected siblings from 1 family (PMID: 21605996, 19862843, 22704482, 1898413, 17723315, 17027861, 22676651, 25673129, 20033296, 20308911), and has been identified in 0.003% (1/30616) of South Asian chromosomes by gnomAD by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs121907937). Although this variant has been seen in the general population, its frequency is low enough to be consistent with a recessive carrier frequency. This variant has also been reported likely pathogenic by Counsyl and pathogenic by OMIM in ClinVar (Variation ID: 4022). Another variant at this position, p.Glu521Val, has been reported as a VUS in association with disease in the literature (PMID: 25526786). In vitro functional studies, including transfection of COS cells and a Western Blot, provide some evidence that the p.Glu521Lys variant may impact protein processing and function (PMID: 1898413, 19862843). However, these types of assays may not accurately represent biological function. Computational prediction tools and conservation analyses suggest that this variant may impact the protein, though this information is not predictive enough to determine pathogenicity. The presence of this variant in combination with reported pathogenic variants in compound heterozygous individuals with Glycogen Storage Disease II increases the likelihood that the p.Glu521Lys variant is pathogenic (PMID: 25673129, 17723315, 20033296). The phenotype of homozygous and heterozygous individuals with this variant is highly specific for Glycogen Storage Disease II based on GAA enzyme activity assays (PMID: 21605996, 25673129, 22676651, 20033296, 17723315, 1898413). In summary, this variant meets criteria to be classified as pathogenic for Glycogen Storage Disease II in an autosomal recessive manner based on in vitro functional studies and multiple occurrences with pathogenic variants in affected individuals. ACMG/AMP Criteria applied: PM3, PS3, PM2, PP3, PP4 (Richards 2015).

The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: <0.001%). The variant is in trans with NM_000152.5:c.1556T>C variant. In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.93; 3Cnet: 0.95). Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000004022). The variant is in trans with the other variant. Different missense changes at the same codon (p.Glu521Gln, p.Glu521Val) have been reported to be associated with GAA related disorder (ClinVar ID: VCV000972760 / PMID: 18425781, 25526786). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

Variant summary: GAA c.1561G>A (p.Glu521Lys) results in a conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 4e-06 in 251268 control chromosomes. c.1561G>A has been reported in the literature in multiple individuals affected with Glycogen Storage Disease, Type 2 (Pompe Disease) in the homozygous and compound heterozygous state (eg. Hermans_1991, Herzog_2012, Liu_2014, Montagnese_2016, etc). These data indicate that the variant is very likely to be associated with disease. The variant has been reported to have <10% enzyme activity in cell culture experiments (Hermans_1991, Flanagan_2009). Two clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

The c.1561G>A variant in GAA is a missense variant predicted to cause substitution of glutamic acid to lysine at amino acid 521. This variant is rare in the general population with a frequency below the threshold expected for the associated phenotype(s). This variant has been observed in one or more individuals affected with the associated recessive disease, as either homozygous or compound heterozygous with a second variant (PMID: 17723315, 22676651). Functional studies show that this variant may disrupt protein function (PMID: 19862843). Given the available evidence, this variant is classified as Pathogenic.

GAA: PM3:Strong, PM1, PM2, PM5, PP3

This sequence change replaces glutamic acid, which is acidic and polar, with lysine, which is basic and polar, at codon 521 of the GAA protein (p.Glu521Lys). This variant is present in population databases (rs121907937, gnomAD 0.003%). This missense change has been observed in individual(s) with clinical features of Pompe disease (PMID: 1898413, 17723315). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 4022). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt GAA protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects GAA function (PMID: 1898413). For these reasons, this variant has been classified as Pathogenic.

GAA p.Glu521Lys (c.1561G>A) is a missense variant that changes the amino acid at codon 521 from Glutamic acid to Lysine. This variant has been observed in at least one proband with a GAA-related disorder in the compound heterozygous and/or homozygous state (PMID:37542277;34852371;34734785;33301762;1898413;17723315;20033296;25526786). At least one functional study has demonstrated a substantial alteration in protein function relative to the wild-type (PMID:1898413;19862843). It is absent or not present at a significant frequency in gnomAD. In silico models predict that this variant is possibly or probably damaging. In conclusion, we classify GAA p.Glu521Lys (c.1561G>A) as a pathogenic variant.

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