This variant, c.525delT (p.Glu176ArgfsTer45), is one of the most common variants reported in individuals with Pompe disease; over 70 patients are listed in the Erasmus database (http://www.pompevariantdatabase.nl/). It is a frameshift variant that is predicted to result in a premature termination codon, nonsense mediated decay, and lack of gene product, meeting PVS1. This is supported by the finding of c.525delT in individuals with no GAA cross-reactive immunological material in cultured skin fibroblasts i.e. CRIM-negative (PMID 22252923, 31342611), no detectable increase in GAA activity or GAA protein when cDNA with the variant was expressed in COS cells (PMID 7881422), and low expression of all GAA exons based on qRT-PCR data from a homozygous patient (PMID 25243733). The highest population minor allele frequency in gnomAD v2.1.1 is 0.000188 in the European non-Finnish population, meeting PM2. Thirty patients meeting the ClinGen LSD VCEP's specifications for PP4 are listed here (PMIDs 8558570, 24590251, 25243733, 26497565, 27142047, 29422078) and include patients who are homozygous for the variant, or compound heterozygous for the variant and either c.-32-13T>G, c.2481+110_2646+39del (exon 18 deletion), c.1802C>A (p.Ser601Ter), c.2608C>T (p.Arg870Ter), c.1548G>A (p.Trp516Ter), c.2237G>A (p.Trp746Ter), and c.670C>T (p.Arg224Trp). The maximum strength for PM3 (PM3_VeryStrong) was applied. There is a ClinVar entry for this variant (Variation ID: 4033, 2 star review status) with 8 laboratory submitters classifying the variant as pathogenic. In summary, this variant meets the criteria to be classified as pathogenic for Pompe disease. GAA-specific ACMG/AMP criteria applied, as specified by the ClinGen LSD VCEP: PVS1, PM2, PM3_Very Strong, PP4.

Variant summary: The GAA c.525delT (p.Glu176ArgfsX45) variant results in a premature termination codon, predicted to cause a truncated or absent GAA protein due to nonsense mediated decay, which are commonly known mechanisms for disease. This variant was found in 8/113628 control chromosomes at a frequency of 0.0000704, which does not exceed the estimated maximal expected allele frequency of a pathogenic GAA variant (0.0042205). The variant has been reported in multiple affected individuals via publications and showed abolished to very little activity. In addition, multiple clinical diagnostic laboratories/reputable databases classified this variant as pathogenic. Taken together, this variant is classified as pathogenic.

The GAA c.525delT; p.Glu176fs variant (rs386834235) is reported in the literature in the homozygous or compound heterozygous in multiple individuals affected with glycogen storage disease type II, also called Pompe disease (Hermans 1994, Kroos 1995, Remiche 2014, Wens 2012). This variant is found in the general population with an overall allele frequency of 0.01% (27/271120 alleles) in the Genome Aggregation Database. This variant causes a frameshift by deleting a single nucleotide, so it is predicted to result in a truncated protein or mRNA subject to nonsense-mediated decay. Functional studies suggest that cells expressing this variant exhibit no measurable alpha-glucosidase catalytic activity (Hermans 1994). Based on available information, this variant is considered to be pathogenic. References: Hermans MM et al. The effect of a single base pair deletion (delta T525) and a C1634T missense mutation (pro545leu) on the expression of lysosomal alpha-glucosidase in patients with glycogen storage disease type II. Hum Mol Genet. 1994 Dec;3(12):2213-8. Kroos MA et al. Glycogen storage disease type II: frequency of three common mutant alleles and their associated clinical phenotypes studied in 121 patients. J Med Genet. 1995 Oct;32(10):836-7. Remiche G et al. Extended phenotype description and new molecular findings in late onset glycogen storage disease type II: a northern Italy population study and review of the literature. J Neurol. 2014 Jan;261(1):83-97. Wens SC et al. Remarkably low fibroblast acid a-glucosidase activity in three adults with Pompe disease. Mol Genet Metab. 2012 Nov;107(3):485-9.

This sequence change is a single base pair deletion in exon 2, c.525del, that results in an amino acid frameshift and the creation of a premature stop codon 44 amino acids downstream of the change, p.Glu176Argfs*45. This sequence change is predicted to result in an abnormal, truncated GAA protein that is likely to affect its normal function. The p.Glu176Argfs*45 change has been reported in several individuals with Glycogen storage disease II and is a known pathogenic variant common in the Dutch population (PMIDs: 8558570, 14695532, 21439876, 22676651, 24158270). This sequence change has been described in the gnomAD database with a relatively low frequency of 0.019% in the European sub-population. Based on these evidence, the c.525del variant is classified as pathogenic.

The p.Glu176ArgfsTer45 variant in GAA has been reported in at least 77 individuals (including 10 Italian, 6 Dutch, 5 from the UK, 2 Australian, 1 German, and 1 Brazilian individuals) with Glycogen Storage Disease II, segregated with disease in 7 affected relatives from 3 families (PMID: 17723315, 7881422, 23000108, 22980766, 12923862, 16917947, 25243733, 18429042, 14695532, 8990003, 18607768, 19588081, 26497565, 24158270, 27189384, 8558570, 27142047, 24590251, 23430912), and has also been reported pathogenic (by EGL, GeneDx, Illumina, Counsyl, Invitae, Mayo Clinic Genetic Testing Laboratories, OMIM, and GeneReviews) in ClinVar (Variation ID: 4033). This variant has been identified in 0.019% (23/122326) of European (non-Finnish) chromosomes and 0.017% (4/23634) of African chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs386834235). Although this variant has been seen in the general population, its frequency is low enough to be consistent with a recessive carrier frequency. In vitro functional studies provide some evidence that the p.Glu176ArgfsTer45 variant may impact GAA expression and activity (PMID: 7881422, 25243733). However, these types of assays may not accurately represent biological function. This variant is predicted to cause a frameshift, which alters the protein's amino acid sequence beginning at position 176 and leads to a premature termination codon 45 amino acids downstream. This alteration is then predicted to lead to a truncated or absent protein. Loss of function of the GAA gene is an established disease mechanism in autosomal recessive Glycogen Storage Disease II. This variant has been seen in homozygous and compound heterozygous patients curated by our study (PMID: 18429042, 14695532, 26497565, 23430912, 17723315, 7881422, 22980766, 16917947, 25243733, 8990003, 18607768, 19588081, 24158270, 27189384, 8558570, 24590251). The phenotype of at least 20 individuals with the variant in the compound heterozygous or homozygous state is highly specific for Glycogen Storage Disease II based on abnormally low GAA activity detected in cultured fibroblasts or muscle cells (PMID: 24158270, 8558570). In summary, this variant meets criteria to be classified as pathogenic for Glycogen Storage Disease II in an autosomal recessive manner based on the predicted impact of the variant and multiple occurrences with pathogenic GAA variants in individuals with Glycogen Storage Disease II. ACMG/AMP Criteria applied: PVS1, PM3, PM2, PP4 (Richards 2015).

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as pathogenic. The following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with glycogen storage disease II (MIM#232300). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0201 - Variant is predicted to cause nonsense-mediated decay (NMD) and loss of protein (premature termination codon is located at least 54 nucleotides upstream of the final exon-exon junction). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (27 heterozygotes, 0 homozygotes). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported in infantile and adolescent patients with glycogen storage disease II (PMID: 7881422, 12923862). This variant has also been reported as pathogenic by more than ten laboratories in ClinVar. (SP) 0701 - Other NMD predicted variants comparable to the one identified in this case have very strong previous evidence for pathogenicity (ClinVar). (SP) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

PP4, PM3_very_strong, PS3, PVS1

The GAA c.525delT p.(Glu176ArgfsTer45) frameshift variant is an established pathogenic variant associated with glycogen storage disease type II. Across a selection of the available literature, it is consistently described as a severe allele and, in functional studies, is associated with significantly reduced mRNA levels, undetectable protein expression and no residual acid alpha glucosidase activity (PMID: 24158270; 25243733; 36246652). Clinically, homozygous individuals typically present with an infantile-onset phenotype whereas compound heterozygous individuals display more variable presentations (PMID: 31086307;31342611). Based on the available evidence, the c.525delT p.(Glu176ArgfsTer45) variant is classified as pathogenic for glycogen storage disease type II.

This sequence change creates a premature translational stop signal (p.Glu176Argfs*45) in the GAA gene. It is expected to result in an absent or disrupted protein product. Loss-of-function variants in GAA are known to be pathogenic (PMID: 18425781, 22252923). This variant is present in population databases (rs386834235, gnomAD 0.02%). This premature translational stop signal has been observed in individual(s) with Pompe disease (PMID: 8558570, 14695532, 18429042, 21439876, 22676651, 24158270, 25243733). It is commonly reported in individuals of Dutch ancestry (PMID: 8558570, 14695532, 18429042, 21439876, 22676651, 24158270, 25243733). ClinVar contains an entry for this variant (Variation ID: 4033). For these reasons, this variant has been classified as Pathogenic.

GAA: PM3:Very Strong, PVS1, PM2

NM_000152.3(GAA):c.525delT(E176Rfs*45) is classified as pathogenic in the context of Pompe disease. Sources cited for classification include the following: PMID 17056254, 16917947, 24158270, 7881422, 8558570, 24590251 and 16702877. Classification of NM_000152.3(GAA):c.525delT(E176Rfs*45) is based on the following criteria: The variant causes a premature termination codon that is expected to be targeted by nonsense-mediated mRNA decay and is reported in individuals with the relevant phenotype. Please note: this variant was assessed in the context of healthy population screening.‚Äã

Expression studies in COS cells show that this variant is associated with a loss of function (Hermans et al., 1994); Frameshift variant predicted to result in protein truncation or nonsense mediated decay in a gene for which loss-of-function is a known mechanism of disease; This variant is associated with the following publications: (PMID: 18607768, 20301438, 16917947, 22676651, 21228398, 22975760, 23000108, 27142047, 14695532, 29946513, 29149851, 30564623, 27189384, 7945303, 9950376, 24715333, 7881422, 8558570, 24590251, 27460347, 27344650, 29422078, 28951071, 18429042, 24158270, 31676142, 31086307, 31589614, 33202836, 8990003, 32528171, 29556838)

ACMG categories: PVS1,PS3,PM2_sup,PM3_strong,PP4

Criteria applied: PVS1,PM3_VSTR,PM2_SUP,PP4

The c.525delT variant in GAA is a frameshift variant predicted to shift the reading frame beginning at codon 176 and leads to a stop codon 45 codons downstream. This variant is expected to result in nonsense mediated decay, truncation, or a dysfunctional protein product. 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: 28763149, 15121988). Given the available evidence, this variant is classified as Pathogenic.

The c.525delT pathogenic mutation, located in coding exon 1 of the GAA gene, results from a deletion of one nucleotide at nucleotide position 525, causing a translational frameshift with a predicted alternate stop codon (p.E176Rfs*45). This variant is reported as a common glycogen storage disease II (Pompe disease) mutation (Reuser AJJ et al. Hum Mutat, 2019 Nov;40:2146-2164). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). This alteration is expected to result in loss of function by premature protein truncation or nonsense-mediated mRNA decay. As such, this alteration is interpreted as a disease-causing mutation.

GAA p.Glu176ArgfsTer45 (c.525del) is a frameshift variant that is predicted to introduce a premature termination codon and result in a truncated or absent protein product. This variant has been observed in at least one proband with a GAA-related disorder in the compound heterozygous and/or homozygous state (PMID:40660301;39273088;38584574;38313679;38250073;38043017;37841659;37765007;37342670;37087815). At least one functional study has demonstrated a substantial alteration in protein function relative to the wild-type (PMID:36246652;7881422). It is absent or not present at a significant frequency in gnomAD. In conclusion, we classify GAA p.Glu176ArgfsTer45 (c.525del) as a pathogenic variant.

PM2_moderate, PVS1_very-strong, PM3_very-strong, PP4_strong