rs143523371
Variant summary
Our verdict is Pathogenic. Variant got 11 ACMG points: 11P and 0B. PM2_SupportingPM3PVS1_StrongPS1_ModeratePP4_Moderate
This summary comes from the ClinGen Evidence Repository: The NM_000152.5:c.546G>A variant alters that last nucleotide of exon 2 of GAA, but does not result in an amino acid change (p.Thr182=). Two different studies, one using real-time RT-PCR to analyze RNA from cultured fibroblasts from an patient with the variant, and another involving minigene analysis showed that the variant disrupts normal splicing, resulting in skipping of exon 2, with a low proportion of normal transcripts produced (PMID:14695532, 31301153). Exon 2 contains the start methionine and signal sequence for GAA (amino acids 1-27; https://www.uniprot.org/uniprot/P10253). In addition, deletion of exon 2 results in complete loss of enzyme activity when expressed in heterologous cells (PMID 7881425; PMID 7717400). As this variant appears to allow a low level of normal splicing to occur, PVS1 was applied at the strong level (PVS1_Strong). Four individuals with a diagnosis of late onset Pompe disease and this variant have been reported with documented laboratory values showing GAA activity in the affected range in dried blood spots or muscle, or <30% activity in skin fibroblasts or <10% activity in muscle and lymphocytes (PMID:14695532, 21484825, 21984055, 25037089) (PP4_Moderate). Two of these patients are compound heterozygous for the variant and another variant in GAA that has been classified as pathogenic by the ClinGen LD VCEP including c.525delT (PMID:14695532), and c.655G>A (p.Gly219Arg) (PMID:25037089), phase not confirmed (PM3). Additional patients have been reported who are compound heterozygous for the variant and either c.736delC (PMID:30564623), c.307T>C (p.Cys103Arg) (PMID:21984055), c.2041-1G>A (PMID:21484825); the allelic data from these patients will be used in the classification of the second variant and is not included here to avoid circular logic. Finally, allelic data for newborn screening cases was not included here due to either lack of confirmation, lack of clinical symptoms in suspected LOPD patients, or presence of pseudodeficiency variants (PMID:33202836, 34995642). The highest population minor allele frequency in gnomAD v2.1.1 is 0.0001 (2/19660 alleles) in the East Asian population, which is lower than the ClinGen LD VCEP’s threshold for PM2_Supporting (<0.001), meeting this criterion (PM2_Supporting). Other variants at the same nucleotide position (c.546G>T, c.546G>C), and in the same splice region (c.546+1G>T, c.546+2T>C, c.546+5G>T, and c.546del2‐5) have been reported in individuals with Pompe disease (https://www.pompevariantdatabase.nl/). All of these variants have been shown to result in skipping of exon 2 using a minigene assay (PMID:31301153). c.546G>T has been classified as pathogenic by the ClinGen Lysosomal Diseases VCEP (PS1_Moderate, PMID:37352859). In summary, this variant meets the criteria to be classified as pathogenic for Pompe disease. GAA-specific ACMG/AMP criteria applied, based in the specification of the ClinGen Lysosomal Diseases VCEP: PVS1_Strong, PS1_Moderate, PM3, PP4_Moderate, PM2_Supporting.(Classification approved by the ClinGen Lysosomal Diseases VCEP on July 18, 2023) LINK:https://erepo.genome.network/evrepo/ui/classification/CA8814895/MONDO:0009290/010
Frequency
Genomes: 𝑓 0.000072 ( 0 hom., cov: 33)
Exomes 𝑓: 0.000092 ( 0 hom. )
Consequence
GAA
NM_000152.5 splice_region, synonymous
NM_000152.5 splice_region, synonymous
Scores
2
Splicing: ADA: 0.9999
2
Clinical Significance
Conservation
PhyloP100: 0.305
Genes affected
GAA (HGNC:4065): (alpha glucosidase) This gene encodes lysosomal alpha-glucosidase, which is essential for the degradation of glycogen to glucose in lysosomes. The encoded preproprotein is proteolytically processed to generate multiple intermediate forms and the mature form of the enzyme. Defects in this gene are the cause of glycogen storage disease II, also known as Pompe's disease, which is an autosomal recessive disorder with a broad clinical spectrum. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2016]
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ACMG classification
Classification made for transcript
Verdict is Pathogenic. Variant got 11 ACMG points.
PVS1
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Transcripts
RefSeq
| Gene | Transcript | HGVSc | HGVSp | Effect | #exon/exons | MANE | UniProt |
|---|---|---|---|---|---|---|---|
| GAA | NM_000152.5 | c.546G>A | p.Thr182= | splice_region_variant, synonymous_variant | 2/20 | ENST00000302262.8 |
Ensembl
| Gene | Transcript | HGVSc | HGVSp | Effect | #exon/exons | TSL | MANE | Appris | UniProt |
|---|---|---|---|---|---|---|---|---|---|
| GAA | ENST00000302262.8 | c.546G>A | p.Thr182= | splice_region_variant, synonymous_variant | 2/20 | 1 | NM_000152.5 | P1 |
Frequencies
GnomAD3 genomes ? AF: 0.0000723 AC: 11AN: 152242Hom.: 0 Cov.: 33
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GnomAD3 exomes AF: 0.0000296 AC: 7AN: 236410Hom.: 0 AF XY: 0.00000769 AC XY: 1AN XY: 130104
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ClinVar
Significance: Pathogenic
Submissions summary: Pathogenic:15
Revision: reviewed by expert panel
LINK: link
Submissions by phenotype
Glycogen storage disease, type II Pathogenic:11
| Pathogenic, reviewed by expert panel | curation | ClinGen Lysosomal Storage Disorder Variant Curation Expert Panel | Jul 18, 2023 | The NM_000152.5:c.546G>A variant alters that last nucleotide of exon 2 of GAA, but does not result in an amino acid change (p.Thr182=). Two different studies, one using real-time RT-PCR to analyze RNA from cultured fibroblasts from an patient with the variant, and another involving minigene analysis showed that the variant disrupts normal splicing, resulting in skipping of exon 2, with a low proportion of normal transcripts produced (PMID: 14695532, 31301153). Exon 2 contains the start methionine and signal sequence for GAA (amino acids 1-27; https://www.uniprot.org/uniprot/P10253). In addition, deletion of exon 2 results in complete loss of enzyme activity when expressed in heterologous cells (PMID 7881425; PMID 7717400). As this variant appears to allow a low level of normal splicing to occur, PVS1 was applied at the strong level (PVS1_Strong). Four individuals with a diagnosis of late onset Pompe disease and this variant have been reported with documented laboratory values showing GAA activity in the affected range in dried blood spots or muscle, or <30% activity in skin fibroblasts or <10% activity in muscle and lymphocytes (PMID: 14695532, 21484825, 21984055, 25037089) (PP4_Moderate). Two of these patients are compound heterozygous for the variant and another variant in GAA that has been classified as pathogenic by the ClinGen LD VCEP including c.525delT (PMID: 14695532), and c.655G>A (p.Gly219Arg) (PMID: 25037089), phase not confirmed (PM3). Additional patients have been reported who are compound heterozygous for the variant and either c.736delC (PMID: 30564623), c.307T>C (p.Cys103Arg) (PMID: 21984055), c.2041-1G>A (PMID: 21484825); the allelic data from these patients will be used in the classification of the second variant and is not included here to avoid circular logic. Finally, allelic data for newborn screening cases was not included here due to either lack of confirmation, lack of clinical symptoms in suspected LOPD patients, or presence of pseudodeficiency variants (PMID: 33202836, 34995642). The highest population minor allele frequency in gnomAD v2.1.1 is 0.0001 (2/19660 alleles) in the East Asian population, which is lower than the ClinGen LD VCEP’s threshold for PM2_Supporting (<0.001), meeting this criterion (PM2_Supporting). Other variants at the same nucleotide position (c.546G>T, c.546G>C), and in the same splice region (c.546+1G>T, c.546+2T>C, c.546+5G>T, and c.546del2‐5) have been reported in individuals with Pompe disease (https://www.pompevariantdatabase.nl/). All of these variants have been shown to result in skipping of exon 2 using a minigene assay (PMID: 31301153). c.546G>T has been classified as pathogenic by the ClinGen Lysosomal Diseases VCEP (PS1_Moderate, PMID: 37352859). In summary, this variant meets the criteria to be classified as pathogenic for Pompe disease. GAA-specific ACMG/AMP criteria applied, based in the specification of the ClinGen Lysosomal Diseases VCEP: PVS1_Strong, PS1_Moderate, PM3, PP4_Moderate, PM2_Supporting. (Classification approved by the ClinGen Lysosomal Diseases VCEP on July 18, 2023) - |
| Pathogenic, criteria provided, single submitter | clinical testing | Illumina Laboratory Services, Illumina | Jan 13, 2022 | The GAA c.546G>A (p.Thr182=) synonymous variant occurs at the last nucleotide of exon 2, and results in substitution of guanine with adenine at position 546 and may result in splicing defects. This variant has been reported in five individuals with late-onset glycogen storage disease, type II, in a compound heterozygous state with a missense, splice site or frameshift variant (Hermans et al. 2004; Bali et al. 2011; Furusawa et al. 2012; Dasouki 2014; Nallamalli et al. 2018). Affected individuals show reduced alpha-glucosidase activity. The highest frequency of this allele in the Genome Aggregation Database is 0.000147 in the European (non-Finnish) population (version 3.1.2). Another nucleotide change at the same position, c.546G>T, is an established pathogenic variant. Based on the available evidence, c.546G>A (p.Thr182=) variant is classified as pathogenic for glycogen storage disease, type II. - |
| Pathogenic, criteria provided, single submitter | clinical testing | Invitae | Jan 24, 2024 | This sequence change affects codon 182 of the GAA mRNA. It is a 'silent' change, meaning that it does not change the encoded amino acid sequence of the GAA protein. This variant also falls at the last nucleotide of exon 2, which is part of the consensus splice site for this exon. This variant is present in population databases (rs143523371, gnomAD 0.02%). This variant has been observed in individuals with late-onset Pompe disease (PMID: 14695532, 21484825, 25037089). ClinVar contains an entry for this variant (Variation ID: 280955). Studies have shown that this variant alters GAA gene expression (PMID: 14695532). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. This variant disrupts the c.546G nucleotide in the GAA gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 17616415, 19609281, 20202878, 21179066, 21982629, 22196155). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic. - |
| Pathogenic, criteria provided, single submitter | clinical testing | Baylor Genetics | Feb 09, 2024 | - - |
| Likely pathogenic, no assertion criteria provided | clinical testing | Counsyl | Jun 01, 2018 | - - |
| Pathogenic, criteria provided, single submitter | clinical testing | Knight Diagnostic Laboratories, Oregon Health and Sciences University | Mar 30, 2016 | The c.546G>A (p.Thr182=) synonymous/splice variant in the GAA gene has been previously reported in in trans with another pathogenic variant (c.525delT) in one individual affected with a mild, late-onset form of GSDII (Hermans et al., 2004). This variant is located near the canonical splice donor-site in intron 2 (last coding nucleotide in exon 2) of the GAA gene. mRNA expression studies from the affected individual’s fibroblasts showed that this variant reduced gene expression (6.3% relative to control) along with reduced alpha-glucosidase activity (3% relative to control); however, abnormally spliced transcripts were not observed (Hermans et al., 2004). Other single nucleotide variants at the same coding position (c.546) have been reported in affected individuals displaying a milder phenotype (Gort et al., 2007; Maimaiti et al., 2009; Kobayashi et al., 2010; Shimada et al., 2011). Furthermore, Tsuburaya RS et al., (2012), identified a c.546G>T variant in the homozygous state in two individuals with adult-onset Pompe disease and showed, by RNA splicing studies, that this variant resulted in exon 2 skipping and reduced enzymatic activity (7.5% and 12.3% respectively). The c.546G>A variant is reported at low frequency in the population databases (Exome Sequencing Project [ESP] = 0.024%; 1000 Genomes = 0.4%; ExAC = 0.036%). In silico splicing algorithms predict this variant will result in abnormal splicing (Human Splice Finder = Broken WT Donor Site, New ESS Site, ESE Site Broken). Therefore, this collective evidence supports the classification of the c.546G>A (p.Thr182=) as a Pathogenic variant for GSDII; however, variants at this nucleotide position have only been observed in individuals affected with a mild, late-onset form of GSDII. - |
| Pathogenic, no assertion criteria provided | clinical testing | Department of Pathology and Laboratory Medicine, Sinai Health System | - | The GAA p.Thr182= variant was identified in 2 of 136 proband chromosomes (frequency: 0.015) from individuals or families with Glycogen-storage disease type II. Sequence variations studies were included one in silico analysis using three Splice Site Prediction (SSP) programs to evaluate the potential effect on splice sites (MaxEntScan, NNSplice and HBond). The c.546G4A mutation does not lead to an amino-acid substitution but modifies the splice donor site of exon 2 “ACAgtgggc instead of ACGgtgggc” (Hermans_2004, Zampieri_2011). The variant was also identified in dbSNP (ID: rs143523371) as “With Likely Pathogenic allele”; in ClinVar and Cllinvitae databases as Pathogenic by Emory Genetics and Knight Diagnostic Laboratories Oregoon Health and Sciences University. In addition, the variant was identified in the 1000 Genomes Project in 1 of 5000 chromosomes (frequency: 0.0002) and in the NHLBI GO Exome Sequencing Project in 1 of 4134 African American alleles (Freq: 0.0002). The variant was not identified in the GeneInsight-COGR, Cosmic, MutDB, databases. The variant was identified in control databases in 7 of 263174 chromosomes at a frequency of 0.000027 (Genome Aggregation Consortium Feb 27, 2017). The c.546G>A variant occurs in the last base of the exon. This position has been shown to be part of the splicing consensus sequence and variants involving this position sometimes affect splicing. In addition, 3 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing. In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic. - |
| Likely pathogenic, criteria provided, single submitter | curation | Broad Center for Mendelian Genomics, Broad Institute of MIT and Harvard | Jan 22, 2020 | The heterozygous c.546G>A (p.Thr182=) variant in GAA has been reported in 4 individuals (including 1 British individual) with Glycogen Storage Disease II (PMID: 21984055, 21484825, 14695532, 25037089), and has also been reported pathogenic (by Invitae, EGL Genetic Diagnostics, Oregon Health and Sciences University, and Integrated Genetics) and likely pathogenic (by Counsyl) in ClinVar (Variation ID: 280955). This variant has been identified in 0.010% (2/19660) of East Asian chromosomes, 0.008503% (2/23522) of African chromosomes, and 0.003% (4/121752) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs143523371). 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 is located in the last three bases of the exon, which is part of the 5' splice region. Computational tools do suggest an impact to splicing. The Threonine (Thr) at position 182 is not well conserved in mammals or evolutionary distant species, raising the possibility that a change at this position may be tolerated. However, this information is not predictive enough to rule out pathogenicity. In vitro functional studies (e.g. RT-PCR) provide some evidence that the c.546G>A variant may significantly reduce GAA expression but may not cause abnormal splicing (PMID: 14695532). However, these types of assays may not accurately represent biological function. The presence of this variant in combination with 2 pathogenic variants and in individuals with Glycogen Storage Disease II increases the likelihood that the c.546G>C variant is pathogenic (PMID: 21484825, 25037089). The phenotype of at least two individuals heterozygous for this variant is highly specific for Glycogen Storage Disease II with reduced GAA activity detected in relevant tissue (PMID: 21484825, 25037089). Two additional variants at the same position, c.546G>T and c.546G>C, have been reported as a VUS or likely pathogenic in association with Glycogen Storage Disease II in ClinVar (Variation ID: 370637, 281056). In summary, although additional studies are required to fully establish its clinical significance, this variant is likely pathogenic. ACMG/AMP Criteria applied: PM2, PM3, PP3, PP4, PS3_Supporting (Richards 2015). - |
| Pathogenic, criteria provided, single submitter | clinical testing | Women's Health and Genetics/Laboratory Corporation of America, LabCorp | Mar 02, 2022 | Variant summary: GAA c.546G>A (p.Thr182Thr) alters a non-conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Four predict the variant abolishes a canonical 5 prime splicing donor site. Four predict the variant creates a cryptic 3 prime acceptor site. Goina_2019 have demonstarted that c.546G>A, c.546G>C and c.546G>T leads to exon 2 skipping compared to wild type. This is consistent with c.546G appears to be a mutational hot-spot, as other alterations of this nucleotide, c.546G>T and c.546G>C, leading to the same synonymous change (p.Thr182=) are associated with glycogen storage disease in HGMD. The variant allele was found at a frequency of 3e-05 in 236410 control chromosomes (gnomAD). c.546G>A has been reported in the literature in individuals affected with Glycogen Storage Disease, Type 2 (Pompe Disease)(examples: Bali_2011, Hermans_2004 and Ficicioglu_2020). These data indicate that the variant is associated with disease. Eight clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 and classified the variant as pathogenic (n=6) and likely pathogenic (n=2). Based on the evidence outlined above, the variant was classified as pathogenic. - |
| Pathogenic, no assertion criteria provided | clinical testing | Natera, Inc. | Sep 16, 2020 | - - |
| Pathogenic, criteria provided, single submitter | clinical testing | Fulgent Genetics, Fulgent Genetics | Oct 16, 2021 | - - |
not provided Pathogenic:3
| Pathogenic, criteria provided, single submitter | clinical testing | GeneDx | Jun 02, 2022 | Published real-time RT-PCR studies showed that mRNA containing the c.546 G>A variant was reduced to 6.3% of normal mRNA levels, which likely corresponded to the greatly reduced enzyme activity in proband fibroblasts (Hermans et al., 2004); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports a deleterious effect on splicing; This variant is associated with the following publications: (PMID: 21179066, 25525159, 14695532, 31301153, 21484825, 30564623, 31980526, 25037089, 33202836) - |
| Pathogenic, criteria provided, single submitter | clinical testing | Eurofins Ntd Llc (ga) | Nov 22, 2017 | - - |
| Pathogenic, criteria provided, single submitter | clinical testing | Revvity Omics, Revvity | Nov 06, 2023 | - - |
Glycogen storage disease Pathogenic:1
| Likely pathogenic, criteria provided, single submitter | clinical testing | Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine | May 15, 2017 | The c.546G>A (p.Thr182Thr) variant in GAA has been reported in 1 compound heterozygous individual with late-onset glycogen storage disease type II. In vitro mRNA studies showed reduced enzyme activity compared to controls, and a different cDNA change at this position (c.546G>T; p.Thr182Thr) has been previously reported to alter splicing and enzyme activity in at least 3 individuals with glycogen storage disease type II (Hermans 2004, Maimaiti 2009, Shimada 2011). This variant has also been reported in ClinVar (280955). The c.546G>A (p.Thr182Thr) variant has been identified in 3/119890 European chromosomes by the genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/; dbSNP rs143523371). 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 is located in the last three bases of the exon, which is part of the 5' splice region. Computational tools suggest an impact to splicing. However, this information is not predictive enough to determine pathogenicity. Loss of function of the GAA gene is an established disease mechanism in autosomal recessive glycogen storage disease, and the presence of this variant in combination with a reported pathogenic variant in an individual with glycogen storage disease type II increases the likelihood that this variant is pathogenic. In summary, although additional studies are required to fully establish its clinical significance, the c.546G>A (p.Thr182Thr) variant is likely pathogenic. - |
Computational scores
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Benign
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dbscSNV1_ADA
Pathogenic
dbscSNV1_RF
Pathogenic
SpliceAI score (max)
Details are displayed if max score is > 0.2
DS_DL_spliceai
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Find out detailed SpliceAI scores and Pangolin per-transcript scores at