The NM_000152.5:c.1441T>C variant in GAA is predicted to result in the missense substitution of tryptophan by arginine at amino acid 481 (p.Trp481Arg). The variant has been reported in at least 6 individuals with Pompe disease in compound heterozygosity with another variant in GAA that has been classified as pathogenic by the ClinGen Lysosomal Diseases VCEP. It was confirmed in trans with c.1326+1G>A (PMIDs: 10189220, 9862843), and was found in unconfirmed phase with c.-32-13T>G (PMID: 14695532, 18757064, 22676651, 27189384, 29556838, 34357340); c.2560C>T (p.Arg854Ter) (PMIDs: 31086307, 35787971), c.1979G>A (p.Arg660His) (PMID: 31086307), and c.2481+102_c.2646+31del (PMID: 26497565, 28657663). Another patient is compound heterozygous for the variant and c.1556T>C (p.Met519Thr) (PMID: 31086307); the allelic data from this patient will be used in the assessment of the other variant and is not included here to avoid circular logic (PM3_Strong). This variant has been reported in individuals with specific phenotypic features of Pompe disease including documented laboratory values revealing GAA deficiency, individuals on enzyme replacement therapy, patients with documented symptoms of infantile onset Pompe disease, and elevated urine Hex4 (PMID: 26497565, 28657663, 31086307, 34357340) (PP4_Moderate). Trp481 is a residue that crystallography studies have shown to be important in the architecture of the active site and substrate binding of GAA; this residues has, therefore, has been defined as a critical residue by the ClinGen Lysosomal Diseases VCEP (https://www.biorxiv.org/content/10.1101/212837v1.full.pdf, PMID: 29061980) (PM1). In two independent studies, expression of the variant in COS cells results in <2% GAA activity compared to normal, but also a significant amount of mature GAA protein, suggesting that the variant impacts catalysis rather than protein production or stability (PMID: 14695532, 19862843). The computational predictor REVEL gives a score of 0.967 which is above the threshold of 0.7, evidence that correlates with impact to GAA function (PP3). There is a ClinVar entry for this variant (Variation ID: ). In summary, this variant meets the criteria to be classified as pathogenic for Pompe disease. GAA-specific ACMG/AMP criteria met, as specified by the ClinGen Lysosomal Diseases Variant Curation Expert Panel (Specifications Version 2.0): PM3_Strong, PM1, PP4_Moderate, PP3, PS3_Supporting, PM2_Supporting, (Classification approved by the ClinGen Lysosomal Diseases VCEP on Oct. 1, 2023)

Variant summary: GAA c.1441T>C (p.Trp481Arg) results in a non-conservative amino acid change in the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 2.2e-05 in 276888 control chromosomes. c.1441T>C has been reported in the literature in individuals affected with Glycogen Storage Disease, Type 2 (Pompe Disease), with reported severely deficient GAA enzyme activity (Tsai_2017, Hermans_2004, Herzog_2012, Raben_1998, van Capelle2016). These data indicate that the variant is likely to be associated with disease. No clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014. Based on the evidence outlined above, the variant was classified as pathogenic.

Not observed at a significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 31254424, 19862843, 21228398, 28657663, 22676651, 18757064, 15501829, 16580018, 31086307, 10189220, 14695532, 26497565, 27189384, 33202836, 29556838, 27535533, 22253258, 19343043)

The GAA c.1441T>C; p.Trp481Arg variant (rs772883420) is reported in the literature in individuals with glycogen storage disease who carry an additional pathogenic GAA variant (Raben 1999, Tsai 2017, van der Meijden 2018). The p.Trp481Arg variant is also reported in ClinVar (Variation ID: 189007). It is observed in the general population with an overall allele frequency of 0.002% (6/282252 alleles) in the Genome Aggregation Database (v2.1.1). Computational analyses predict that this variant is deleterious (REVEL: 0.967). In support of these predictions, functional characterization of the variant protein shows altered function (Flanagan 2009, Hermans 2004). Based on available information, this variant is considered to be likely pathogenic. References: Flanagan JJ et al. The pharmacological chaperone 1-deoxynojirimycin increases the activity and lysosomal trafficking of multiple mutant forms of acid alpha-glucosidase. Hum Mutat. 2009 Dec;30(12):1683-92. PMID: 19862843. Hermans MM et al. Twenty-two novel mutations in the lysosomal alpha-glucosidase gene (GAA) underscore the genotype-phenotype correlation in glycogen storage disease type II. Hum Mutat. 2004 Jan;23(1):47-56. PMID: 14695532. Raben N et al. Lee E, Lee L, Hirschhorn R, Plotz PH. Novel mutations in African American patients with glycogen storage disease Type II. Mutations in brief no. 209. Online. Hum Mutat. 1999;13(1):83-4. PMID: 10189220. Tsai AC et al. Next generation deep sequencing corrects diagnostic pitfalls of traditional molecular approach in a patient with prenatal onset of Pompe disease. Am J Med Genet A. 2017 Sep;173(9):2500-2504. PMID: 28657663. van der Meijden JC et al. Long-term follow-up of 17 patients with childhood Pompe disease treated with enzyme replacement therapy. J Inherit Metab Dis. 2018 Nov;41(6):1205-1214. PMID: 29556838.

The p.W481R pathogenic mutation (also known as c.1441T>C), located in coding exon 9 of the GAA gene, results from a T to C substitution at nucleotide position 1441. The tryptophan at codon 481 is replaced by arginine, an amino acid with dissimilar properties. This variant has been identified in the homozygous state and/or in conjunction with other GAA variant(s) in individual(s) with features consistent with glycogen storage disease type II (Raben N et al. Hum Mutat, 1999;13:83-4; Hermans MM et al. Hum Mutat, 2004 Jan;23:47-56; Flanagan JJ et al. Hum Mutat, 2009 Dec;30:1683-92; Broomfield A et al. J Inherit Metab Dis, 2016 Mar;39:261-71; Tsai AC et al. Am J Med Genet A, 2017 Sep;173:2500-2504; Desai AK et al. Mol Genet Metab Rep, 2019 Sep;20:100475; Ficicioglu C et al. Int J Neonatal Screen, 2020 Nov;6:[ePub ahead of print]; Viamonte MA et al. J Hum Genet, 2021 Nov;66:1089-1099; Zhang T et al. Mol Genet Metab, 2022 Aug;136:296-305). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the supporting evidence, this variant is interpreted as a disease-causing mutation.

This sequence change replaces tryptophan, which is neutral and slightly polar, with arginine, which is basic and polar, at codon 481 of the GAA protein (p.Trp481Arg). This variant is present in population databases (rs772883420, gnomAD 0.008%). This missense change has been observed in individual(s) with Pompe disease (PMID: 18757064; internal data). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. ClinVar contains an entry for this variant (Variation ID: 189007). Invitae Evidence Modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) 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: 14695532, 19862843). For these reasons, this variant has been classified as Pathogenic.

GAA p.Trp481Arg (c.1441T>C) is a missense variant that changes the amino acid at codon 481 from Tryptophan to Arginine. This variant has been observed in at least one proband with a GAA-related disorder in the compound heterozygous and/or homozygous state (PMID:40562809;35787971;34357340;31193175;26497565;28657663;27189384;22676651). At least one functional study has demonstrated a substantial alteration in protein function relative to the wild-type (PMID:14695532;19862843). The variant is located in a mutational hotspot and/or important functional domain. 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.Trp481Arg (c.1441T>C) as a pathogenic variant.

The p.Trp481Arg variant in GAA has been reported in 6 individuals (including 1 African American and 1 German individuals) with Glycogen Storage Disease II (PMID: 27189384, 18757064, 10189220, 19862843, 26497565, 22676651, 14695532, 28657663), and has also been reported likely pathogenic by Counsyl and pathogenic by Invitae and Integrated Genetics in ClinVar (Variation ID: 189007). This variant has been identified in 0.008% (2/24904) of African chromosomes and 0.003% (4/128730) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs772883420). 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.Trp481Arg variant may impact ligand binding and GAA activity but not GAA levels (PMID: 19862843, 14695532, 15501829). 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 pathogenic variants and in individuals with Glycogen Storage Disease II increases the likelihood that the p.Trp481Arg variant is pathogenic (PMID: 26497565, 22676651, 27189384, 28657663). The phenotype of individuals heterozygous for this variant is highly specific for Glycogen Storage Disease II based on very low GAA activity in assays with relevant tissue (PMID: 26497565, 22676651, 27189384, 28657663). In summary, this variant meets criteria to be classified as pathogenic for p.Trp481Arg in an autosomal recessive manner based on in vitro functional evidence and multiple occurrences with pathogenic variants in individuals with Glycogen Storage Disease II. ACMG/AMP Criteria applied: PS3, PM2, PM3, PP3, PP4 (Richards 2015).

PP3_strong, PP4_moderate, PM3_strong, PS3_supporting

The c.1441T>C variant in GAA is a missense variant predicted to cause substitution of tryptophan to arginine at amino acid 481. 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: 31086307). This variant is located in a functionally critical region of the protein. Given the available evidence, this variant is classified as Pathogenic.

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