ClinVar Genomic variation as it relates to human health

The NM_000152.5:c.-32-13T>G variant occurs in the acceptor splice site region of intron 1 of GAA and results in a reduced level of normally spliced GAA transcripts (“leaky splicing”) allowing for the production of residual GAA activity. This variant, historically termed IVS1-13T>G and -45T>G, is the most common GAA variant identified in patients with late-onset Pompe disease; about 36-90% of individuals with late-onset Pompe disease are heterozygous for this variant (PMID: 20301438, 31342611). Haplotype analysis suggests that c.-32-13T>G has arisen at least twice (PMID: 17210890). RT-PCR analysis of fibroblast RNA from patients with the variant, in addition to minigene studies assessing the splicing impact of c.-32-13T>G, have identified various aberrantly spliced transcripts as well as a low level of the normal transcript. The aberrant splice variants include SV1, which retains the first 36 nt of intron 1 and lacks exon 2; SV2, in which exon 2 is completely spliced out; and SV3, in which exon 2 is partially spliced out. The same aberrant splicing variants, missing all or part of exon 2, have also been found at low levels in the RNA of unaffected individuals and when the normal GAA sequence is analyzed in minigene studies (PMID: 7717400, 8817337, 17210890, 24150945). Overall, the variant appears to reduce the level of normal GAA transcripts to about 10-20% of wild type. This “leaky splicing” allows for some active GAA enzyme to be produced, thereby delaying onset of symptoms in individuals who carry this variant. It should be noted that these in vitro observations pertain to data collected in non muscle cell lines, the precise effect that this variant may have in vivo within muscle is not known. In cells from patients and cells transfected with the variant, antisense oligonucleotides and other treatments have been shown to increase the level of exon 2 inclusion, producing correctly spliced transcripts; to increase the residual activity of GAA; and to reduce the level of glycogen (PMID: 24150945, 28624228, 28629821, 31301153, 32317649, 33426149, 36401034). Based on the accumulation of evidence, PVS1 is applied at strong (PMID: 37352859). Hundreds of patients with this variant have been reported in the literature (see list of publications in https://www.pompevariantdatabase.nl/ ). There are numerous of examples of affected patients who are heterozygous for c.-32-13T>G and another variant in GAA that has been classified as pathogenic by the ClinGen LD GCEP (pathogenic classification does not require use of c.-32-13T>G for PM3). To provide some examples, the variant has been found in compound heterozygosity with c.525delT (ClinVar Variation ID: 4033, SCV001443331.1) (4 probands; max 2 x 0.5 points) (PMID: 27189384), c.1051delG (ClinVar Variation ID: 188841, SCV001371764.1) (0.5 points) (PMID: 27189384), c.2481+102_2646+31del (ClinVar Variation ID: 657307) (0.5 points) (PMID: 27189384), c.2331+2T>A (ClinGen Variation ID: 371281) (2 probands) (2 x 0.5 points) (PMID: 27189384), c.1548G>A (p.Trp516Ter) (ClinVar Variation ID: 189025, SCV001371740.1) (0.5 points) (PMID: 27189384), c.1441T>C (p.Trp481Arg) (ClinVar Variation ID: 189007, SCV004227917.1) (0.5 points) (PMID: 27189384), c.1551+1G>C (ClinVar Variation ID: 554983) (0.5 points) (PMID: 7881425), c.784G>A (p.Glu262Lys) (ClinVar Variation ID: 188806, SCV002032128.1) (0.5 points) (PMID: 25673129), and c.670C>T (p.Arg224Trp) (Variation ID: 189188, SCV001371775.1) (0.5 points) (PMID: 25673129), as well as homozygous individuals (max 2 x 0.5 points) (PMID: 25673129, 26231297 34405923). >7 points (PM3_VeryStrong). While the variant has never been associated with classical infantile-onset Pompe disease, the age of onset and severity of disease in patients with the variant can vary widely, even in individuals who are compound heterozygous for c.-32-13T>G and a complete loss of function variant (PMID: 17210890, 34405923). Clinically affected patients who are homozygous for the variant have also been reported. However, some homozygous individuals appear to remain asymptomatic, leading to discussions on how best to follow these individuals if identified on newborn screen (PMID: 30922962). Studies indicate that one of the factors that can modify the phenotype is the presence of another variant, c.510C>T, in cis with c.-32-13T>G. This variant has been shown to further reduce the level of normal GAA mRNA causing early onset and greater severity of symptoms (PMID: 30922962). The highest minor allele frequency (MAF) in gnomAD v2.1.1. is 0.005293 (614/116004 alleles) in the European non-Finnish population, with one homozygote in the “remaining individuals” group. In gnomAD v4.1.0, the highest population MAF is 0.006355 (7335/1154180 alleles; 19 homozygotes) in the European non-Finnish population. Although this allele frequency meets the BS1 threshold suggested by the LD VCEP, the fact that this variant is the most common variant in individuals with late onset Pompe disease accounts for the population based allele frequency that is observed. Therefore, BS1 was not applied. In addition there is abundant evidence to support its pathogenicity. Other variants in the same splice region have been identified in patients with Pompe disease including c.-32-3C>G, c.-32-3C>A, c.-32-2A>G, and c.-32-1G>C (PMID: 31301153, 33560568). The classification of c.-32-13T>G will be used to support the classification of these other variants. Therefore, to avoid circular logic, their classification was not used in the classification of c.-32-13T>G. There is a ClinVar entry for this variant (Variation ID: 4027). In summary, the c.-32-13T>G variant (also known as IVS1-13T>G) meets the criteria to be classified as pathogenic for Pompe disease. It results in residual GAA activity and is the most common GAA variant causing late-onset Pompe disease. GAA-specific ACMG/AMP criteria met, as specified by the ClinGen Lysosomal Diseases Variant Curation Expert Panel (Specifications version 2.0): PVS1_Strong, PM3_Very Strong, PP4_Moderate. (Classification approved by the ClinGen Lysosomal Diseases Variant Curation Expert Panel on October 1st, 2024)

c.-32-13T>G, an intronic variant, accounts for 36% - 90% of late-onset Pompe disease (GeneReviews: Leslie and Tinkle, update 2013). It has been reported in trans with a pathogenic variant and segregates with disease in multiple affected individuals in several families (Kroos et al. 2007). Functional studies have shown that this variant affect splicing and causes reduction in the enzyme activity (Boerkoel CF et al., 1995; Kroos et al. 200). A reputable clinical diagnostic laboratory (Emory Genetics Laboratory) has also classified this variant as pathogenic. Therefore, this collective evidence supports the classification of the c.-32-13T>G as a recessive Pathogenic variant for Glycogen storage storage disease type II.

Variant summary: The GAA variant, c.-32-13T>G, alters a non-conservative nucleotide and 3/5 in silico splicing tools predict this variant to have a moderate effect on splicing pattern consistent with loss of function as the established mechanism of action of disease. These predictions were confirmed by Boerkoel_1995, who showed that this variant leads to exon 2 skipping with a low level of normal mRNA also being transcribed (aka leaky splicing). The leakage of the normal transcript is likely responsible for the remaining low level of GAA activity and late-onset clinical presentation. This variant is attributed to ~70% of late onset Pompe cases, has been reported in trans with several pathogenic variants and segregates with disease in multiple affected individuals in several families. The variant of interest has been reported as Pathogenic by several reputable databases/clinical laboratories dating back from 2007 to present. Taking together, the variant has been classified as "pathogenic."

NM_000152.3(GAA):c.-32-13T>G(aka IVS1-13T>G) is classified as pathogenic in the context of Pompe disease. The variant is seen in 36% to 90% of late-onset Pompe disease and is typically associated with late onset. Sources cited for classification include the following: PMID 24150945, 24158270, 16702877, 21439876, 7881425, 22595200 and 24590251. Classification of NM_000152.3(GAA):c.-32-13T>G(aka IVS1-13T>G) is based on the following criteria: This is a well-established pathogenic variant in the literature that has been observed more frequently in patients with clinical diagnoses than in healthy populations. Please note: this variant was assessed in the context of healthy population screening.

The variant has been reported to be in trans with a pathogenic variant as either compound heterozygous or homozygous in at least 4 similarly affected unrelated individuals (PMID: 24590251, 22613277, 26231297, 24245577, PM3_VS). Functional studies provide strong evidence of the variant having a damaging effect on the gene or gene product (PMID: 7717400, PS3_S). The variant has been reported at least twice as pathogenic/likely pathogenic with clinical assertions and evidence for the classification (ClinVar ID: VCV000004027). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

A heterozygous variant in intron 1 of the GAA gene that affects the position 13 nucleotides upstream to exon 2 was detected. The observed variant c.-32-13T>G has a minor allele frequency of 0.3% and 0.4% in the 1000 genomes and gnomAD database respectively. The variant has been previously reported in patients affected with adult-onset glycogen storage disease type-II (Dardis et al. 2014). The in silico prediction of the variant is damaging by MutationTaster2. The reference codon is conserved across species. In summary, the variant meets our criteria to be classified as a pathogenic variant.

The GAA c.-32-13T>G variant is classified as Likely Pathogenic (PS3_Moderate, PM3_Very_Strong, PP4_moderate) GAA c.-32-13T>G is located in intron 1/19. Functional studies show that the variant results in an increased amount of aberrant splicing and a reduction in normal spliced mRNA. Some residual normal spliced mRNA remains, possibly accounting for the late onset of disease. This variant has been reported as a Class A severity variant (PMID:24510945, 7717400, 18425781) (PS3_moderate). This variant has been detected in trans with a pathogenic variant in affected patients and is considered the most common GAA variant in patients with adult onset Pompe disease (PMID:16917947, 17210890, 27189384, 7881425, 7717400) (PM3_Very strong). The variant has been reported in dbSNP (rs386834236), as Pathogenic by other diagnostic laboratories (ClinVar Variation ID: 4027) and as disease causing in HGMD (CS941489). The clinical features of this case are highly specific for a variant in the GAA gene (PMID:18425781) (PP4_moderate).

The c.-32-13T>G variant in GAA is a well-established pathogenic variant for glycogen storage disease type II (GSD2). This is the most common variant in Caucasian individuals with late-onset GSD2, though it is rarely identified in individuals with the infantile-onset form of the disorder (Kroos 2007 PMID: 17210890, Byrne 2011 PMID: 21439876). This variant segregated with disease in many affected relatives from multiple families (Wens 2013 PMID: 24245577). The c.-32-13T>G variant has also been reported by other clinical laboratories in ClinVar (Variation ID: 4027) and has been identified in 0.55% (45/8122) of Ashkenazi Jewish and in 0.5% (614/116004) of European chromosomes by gnomAD (https://gnomad.broadinstitute.org/). This variant is located in the 3' splice region. Functional studies have demonstrated that this variant alters splicing of the GAA transcript, leading to reduced expression of functional GAA enzyme (Dardis 2014 PMID: 24150945). In summary, despite its high frequency in the general population, the c.-32-13T>G variant meets criteria to be classified as pathogenic for late-onset glycogen storage disease type II in an autosomal recessive manner based upon functional evidence and its identification in numerous affected individuals. ACMG/AMP Criteria applied: PM3_Very Strong, PP1_Strong, PS3_Supporting.

The c.-32-13 T>G pathogenic variant in the GAA gene is a common variant that has been reported in 36-90% of patients with adult-onset GSDII (PMID: 7881425; PMID: 24150945). Functional studies demonstrate that the c.-32-13 T>G mutation results in aberrant gene splicing (PMID: 24150945).

The GAA c.-32-13T>G variant has been reported in at least six studies and is found in a total of 248 individuals with glycogen storage disease type II, including 151 in a compound heterozygous state and 38 in a heterozygous state in whom a second variant in the GAA gene was not found. Zygosity was not specified for 59 of the 248 patients (Huie et al. 1994; Hermans et al. 2004; Montalvo et al. 2006; Kroos et al. 2007; van Capelle et al. 2016; Lukacs et al. 2016). Patients with the c.-32-13T>G variant were found overall to be more severely affected (van Capelle et al. 2016). The variant was absent from 29 controls and is reported at a frequency of 0.00572 in the European American population of the Exome Sequencing Project. Functional studies have shown the presence of the c.-32-13T>G variant causes aberrant splicing, resulting in exclusion of exon 2 in the processed transcript (Huie et al. 1994). Based on the evidence, the c.-32-13T>G variant is classified as pathogenic for glycogen storage disease type II. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

The GAA c.-32-13T>G variant was previously observed in Pompe disease (PMID: 16917947; 17210890; 27189384).

This variant was identified in trans as compound heterozygous with NM_000152.5:c.307T>G.

The variant c.-32-13T>G in the GAA gene is reported as pathogenic for glycogen storage disease type 2 (Pompe disease) in ClinVar (Variation ID: 4027) and as pathogenic in the Global Variome shared LOVD database v.3.0. The variant was firstly identified by Huie et al., 1994 (PMID: 7881425) in a patient with late onset Pompe disease. Subsequent functional studies have clarified that this variant alters the splicing process, leading to the production of a transcript with exon 2 deletion, although a low amount of normal transcript is produced, which may explain the role of this variant in late forms of the disease (Boerkoel et al., 1995, PMID: 7717400; Dardis et al., 2014, PMID: 24150945). This variant represents a common mutation that is present in approximately 40% -70% of alleles in patients with late forms (PMID: 24150945). According to Leslie et Bailey, 2017 (PMID: 20301438) the variant is observed in 36% to 90% of late-onset Pompe disease cases. The variant is reported with an estimated allelic frequency of 0.003445 in gnomAD exomes and 0.003094 in gnomAD genomes, with one homozygous individual reported.

The c.-32-13T>G variant is the most common, seen in 36% to 90% of individuals affected with Glycogen storage disease, type II (OMIM: 606800.0006; PMID: 7881425; 7717400; 7668832; 8558570; 11071489; 14695532; 16433701; 16531044; 16917947; 17210890; 17723315; 17643989; 17616415; 18607768; 19588081; 20350966; 21550241; 20559845; 20301438; GeneOne, DASA), and ClinVar contains an entry for this variant (Variation ID: 4027) - PS4; well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product indicates that the variant causes aberrant splicing of the GAA RNA, resulting in most transcripts lacking exon 2 containing the canonical start codon (PMID: 7881425) - PS3; variant detected in trans with a pathogenic variant (PMID: 27649523) - PM3_strong; this variant is present in population databases (rs386834236 - gnomAD 0.376% frequency; ABraOM 0.29% - http://abraom.ib.usp.br/) - BS1; In summary, the currently available evidence indicates that the variant is pathogenic.

The c.-32-13T>G in compound heterocigosity with c.118C>T (p.Arg40Ter) GAA variant has been reported in our laboratory in a 52-year-old woman from England with diagnosis of Pompe disease (onset 10 years before) with parents and four asymptomatic children and alpha-1,4-glucosidase lysosomal enzyme activity study: 0.4 µmol/L/h (cut-off value: >2.0). Pompe Variant Database describes this phenotype in eight patients, all diagnosed between the ages of 20 and 60. It has been previously reported in patients with Pompe disease adult-onset GSDII (PMID: 7881425, 24150945, 16917947; 17210890; 27189384). This variant is present in population databases ( gnomAD allele frequency 0.003401). ClinVar contains an entry for this variant (Variation ID: 4027). Functional studies demonstrate that this variant results in aberrant gene splicing (PMID: 24150945). In summary, c.-32-13T>G GAA variant meets our criteria to be classified as pathogenic for late-onset glycogen storage disease type II in an autosomal recessive manner based upon functional evidence and its identification in numerous affected individuals.

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) 0217 - Non-coding variant with known effect. Experimental studies using patient fibroblasts demonstrated abberant splicing which yields two splicing outcomes, one in which intron 1 is fully spliced out and the other in which 36 nucleotide of intron 1 is retained (PMID: 24150945). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (854 heterozygotes, 1 homozygote). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This is one of the most common pathogenic variants associated with adult-onset Pompe disease (ClinVar) however it has also been reported in patients with early and infantile onset Pompe disease. It has been reported in mostly compound heterozygous but also rarely in homozygous individuals, where incomplete penetrance or environmental factors suggested to affect disease onset (ClinVar, PMID: 26231297, PMID: 17210890). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

Splicing abnormalities, predominantly exon-2 skipping, due to the c.-32-13T>G pathogenic variant were confirmed by RT-PCR.

This sequence change falls in intron 1 of the GAA gene. It does not directly change the encoded amino acid sequence of the GAA protein. RNA analysis indicates that this variant induces altered splicing and is likely to result in the loss of the initiator methionine. This variant is present in population databases (rs386834236, gnomAD 0.6%), including at least one homozygous and/or hemizygous individual. This variant is a well documented cause of late-onset Pompe disease among individuals of European ancestry, and it has also been reported in affected individuals of other ethnicities (PMID: 7881425, 24590251, 21439876, 22613277, 26231297, 24245577). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 4027). Experimental studies have shown that this variant interferes with mRNA splicing of exon 1 of the GAA gene. The effect is such that it allows for some normally spliced transcript to be produced, which is thought to contribute to its role in late-onset as opposed to early-onset Pompe disease (PMID: 7881425, 2510307). For these reasons, this variant has been classified as Pathogenic.

The GAA c.-32-13T>G variant (rs386834236), also known as c.-45T>G, is reported in the literature in multiple individuals affected with adult-onset Pompe disease (Beltran Papsdorf 2014, Boerkoel 1995, Dardis 2014, Golsari 2018, Gort 2007, Kroos 1995, Montalvo 2006, Musumeci 2015, Sacconi 2014). This variant is reported in ClinVar (Variation ID: 4027), and is found in the general population with an overall allele frequency of 0.34% (856/251,700 alleles, including a single homozygote) in the Genome Aggregation Database. This is an intronic variant in a weakly conserved nucleotide, but computational analyses (Alamut v.2.11) predict that this variant may impact splicing by weakening the nearby canonical acceptor splice site. Functional characterization indicates that the variant causes aberrant splicing of the GAA RNA, resulting in most transcripts lacking exon 2 containing the canonical start codon (Boerkoel 1995, Dardis 2014). However, small amounts of full-length transcripts is still generated, resulting in varying amount of residual GAA activity in the patients (Gort 2007, Kroos 1995, Musumeci 2015, Sacconi 2014). Based on available information, the c.-32-13T>G variant is considered to be pathogenic. References: Beltran Papsdorf T et al. Pearls & Oy-sters: clues to the diagnosis of adult-onset acid maltase deficiency. Neurology. 2014 82(9):e73-5. PubMed: 24590251. Boerkoel C et al. Leaky splicing mutation in the acid maltase gene is associated with delayed onset of glycogenosis type II. Am J Hum Genet. 1995 Apr;56(4):887-97. PMID: 7717400. Dardis A et al. Functional characterization of the common c.-32-13T>G mutation of GAA gene: identification of potential therapeutic agents. Nucleic Acids Res. 2014 42(2):1291-302. PubMed: 24150945. Golsari A et al. Prevalence of adult Pompe disease in patients with proximal myopathic syndrome and undiagnosed muscle biopsy. Neuromuscul Disord. 2018 Mar;28(3):257-261. PubMed: 29326002. Gort L et al. Glycogen storage disease type II in Spanish patients: high frequency of c.1076-1G>C mutation. Mol Genet Metab. 2007 92(1-2):183-7. PMID: 17616415. Kross M 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 32(10):836-7. PMID: 17210890. Montalvo A et al. Mutation profile of the GAA gene in 40 Italian patients with late onset glycogen storage disease type II. Hum Mutat. 2006 27(10):999-1006. PMID: 16917947. Musumeci O et al. Homozygosity for the common GAA gene splice site mutation c.-32-13T>G in Pompe disease is associated with the classical adult phenotypical spectrum. Neuromuscul Disord. 2015 25(9):719-24. PMID: 26231297. Sacconi S et al. Atrio-ventricular block requiring pacemaker in patients with late onset Pompe disease. Neuromuscul Disord. 2014 24(7):648-50. PMID: 24844452.

This sequence change falls in intron 1 of GAA. The variant is present in a large population cohort at a frequency of 0.3% (rs386834236, 856/251,700 alleles, 1 homozygote in gnomAD v2.1). It is the most commonly reported variant (homozygous or with a second pathogenic allele) in individuals affected by the late-onset form of glycogen storage disorder type II confirmed by reduced enzyme activities in lymphocytes/muscle tissue, and segregates with disease (PMID: 7881425, 16917947, 26231297). Multiple lines of computational evidence predict no significant splice defect (SpliceAI, MaxEntScan, NNSplice). However, functional assays demonstrate the variant has a leaky splice effect by abrogating binding of splice factors to the exon 2 polypyrimidine tract leading to a significant increase in aberrantly spliced transcripts with some expression of the full-length transcript from the variant allele (PMID: 24150945). Based on the classification scheme RMH Modified ACMG Guidelines v1.3.2, this variant is classified as PATHOGENIC. Following criteria are met: PM3_VeryStrong, PS3_Supporting, PP1, PP4.

The c.-32-13T>G intronic pathogenic mutation (also known as c.-45T>G and IVS1-13T>G) results from a T to G substiution 45 nucleotides before coding exon 1 in the GAA gene. This pathogenic mutation is frequently reported in individuals with adult onset glycogen storage disease type II (Huie ML et al. Hum Mol Genet. 1994;3(12):2231-6). Another study reported this leaky splice site affects the pre-mRNA splicing and binding of regulatory proteins in this region (Dardis A et al. Nucleic Acids Res. 2014;42(2):1291-302). This nucleotide position is not well conserved in available vertebrate species. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Common pathogenic variant reported in 36-90% of individuals with late-onset GSDII (PMID: 7881425, 24150945); Functional studies demonstrate that the c.-32-13 T>G variant results in aberrant gene splicing (PMID: 24150945); This variant is associated with the following publications: (PMID: 24844452, 34530085, 34906502, 34405919, 33807278, 34501319, 33073019, 24590251, 24158270, 22613277, 21439876, 22595200, 25103075, 16917947, 25846667, 26800218, 22975760, 26231297, 27170567, 27460347, 28032299, 27708273, 21109266, 21228398, 26350092, 25673129, 7881425, 29326002, 16531044, 29181627, 28951071, 15986226, 28694071, 21967859, 30827497, 30564623, 30314719, 23417379, 31676142, 32071926, 31980526, 31086307, 30275481, 34440436, 34426522, 28629821, 8990003, 32721234, 32528171, 29556838, 36310651, 37701327, 37342670, 35948506, 32860008, 34020684, 33726816, 33673364, 33188503, 36046397, 35302691, 35741838, 34864681, 35047849, 34852371, 24150945)

GAA: PM3:Very Strong, PM2, PP4, PS3:Supporting

The observed phenotype is merely musculoskeletal. Dyspnea on exertion, difficulty in both genuflection and climbing stairs, progressive muscle weakness in pelvic area, amyotrophy, hyperflexia, early fatigue, myalgias and cramps, Gowers sign were observed in all siblings, while weakness in arms, scapula alata and progresive muscle weakness in scapula area were observed in males only.

This sequence change is an intronic substitution in intron 1, c.-32-13T>G (also known as c.-45T>G). This sequence change has been reported in several individuals with adult-onset glycogen storage disease type II /Pompe disease in both homozygous and compound heterozygous states (PMID: 7881425, 24590251, 21439876, 22613277, 26231297, 24245577). Functional in vivo studies have shown that this sequence change abrogates the binding of the splicing factor U2AF65 to the polypyrimidine tract of exon 2 resulting in aberrant gene splicing (PMID: 24150945). This sequence change has also been termed as a 'leaky splice' variant, as it produces some percentage of the normal amount of messenger RNA which results in a proportional amount of structurally and functionally normal acid a-glucosidase (PMID: 22253258,17210890). This sequence change has been described in the gnomAD database with an overall population frequency of 0.3%. Based on these evidence, the c.-32-13T>G variant is classified as pathogenic.

The GAA c.-32-13T>G variant is located in the 5' untranslated region. This variant has been clearly documented as causative for glycogen storage disease type II when present in trans with another pathogenic GAA variant. The c.-32-13T>G variant is the most common pathogenic variant found in adults with glycogen storage disease type II/late-onset Pompe disease (GSD II/ LOPD) (Huie et al. 1994. PubMed ID: 7881425; Hermans et al. 2004. PubMed ID: 14695532). This variant leads to aberrant splicing of exon 2, which contains the start codon (Dardis et al. 2014. PubMed ID: 24150945). Please note that in the literature, this variant may also be referred to as c.-45T>G or IVS1-13T>G. In summary, this variant is interpreted as pathogenic.

NG_009822.1(NM_000152.3):c.-32-13T>G in the GAA gene has an allele frequency of 0.006 in Ashkenazi Jewish subpopulation in the gnomAD database. Functional studies demonstrate that this variant interferes with mRNA splicing of exon 1 of the GAA gene. Wen ey al reported 22 families with Pompe disease. All carried the most common mutation c.-32-13 T to G in combination with another pathogenic mutation (PMID: 24245577).The patient's phenotype is highly specific for GAA gene (PMID: 7881425; 2510307). Taken together, we interprete this variant as Pathogenic/Likely pathogenic. ACMG/AMP criteria applied: PS3; PS4; PP4.