ClinVar Genomic variation as it relates to human health

The NM_000152.5:c.1327-2A>G variant in GAA occurs within the canonical splice acceptor site of intron 8. It is predicted to cause skipping of biologically-relevant-exon 9 (GAA has 20 exons) resulting in an in-frame deletion of 37 amino acids (amino acids 443-479; ~3.9% of the protein) which forms part of the GAA catalytic barrel (Kroos et al, 2012, PMID 22253258; Deming et al, 2017; DOI 10.1101/212837). As expected, SpliceAI predicts that the variant will obliterate the normal splice acceptor site (score= 0.99). However, SpliceAI also predicts a possible acceptor gain 16 bp upstream from the normal splice site (score= 0.79). If this site were to be used when the normal acceptor site is broken, a frameshift is predicted. However, the report that an individual who is homozygous for this variant and is CRIM-positive is consistent with an in frame deletion (PMID 22252923). PVS1_Strong is applied, based on the specifications of the ClinGen LD VCEP due to predicted loss of an exon that includes a critical region of the protein (PVS1_Strong). At least three probands and a sibling with infantile onset Pompe disease have been reported with this variant and documented GAA activity in the affected range. Two of them were on enzyme replacement therapy; follow up was reported for one patient with clinical improvement (PMID 19067231, 20821053, 31510962). In addition, one individual, identified in a clinical laboratory, is compound heterozygous for the variant and pseudodeficiency variants were shown to be absent (PP4_Moderate). This variant was found in homozygosity in at least two individuals of Emirati origin (PMIDs 19067231, 20821053, 23430803, 24273659, 29122469) (max 2 x 0.5 points allowed for homozygous individuals = 1 point). In addition, an individual has been reported who is compound heterozygous for the variant and another variant in GAA phase unconfirmed, that has been classified as pathogenic by the ClinGen LD VCEP (clinical laboratory data) (0.5 points); and another individual is compound heterozygous for the variant confirmed in trans with c.1437G>C (p.Lys479Asn) (likely pathogenic without use of allelic data from the patient to avoid circular logic) (1 point). Total 2.5 points (PM3_Strong). The highest population minor allele frequency in gnomAD v2.1.1 is 0.0002297 (2/8708 alleles) in the African / African American population, which is lower than the ClinGen LD VCEP's threshold (<0.001) for PM2_Supporting, meeting this criterion (PM2_Supporting). There is a ClinVar entry for this variant (Variation ID: 972762). 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 Lysosomal Diseases Variant Curation Expert Panel (Specifications Version 2.0): PVS1_Strong, PM3_Strong, PP4_Moderate, PM2_Supporting. (Classification approved by the ClinGen Lysosomal Diseases Variant Curation Expert Panel on September 17, 2024).

The c.1327-2A>G variant in GAA has been reported in at least 9 individuals with glycogen storage disease (PMID: 29122469, 24273659, 23430803, 19067231, 18425781) and has been Identified in 0.023% (2/8708) of African chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs1410829147). 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 occurs in the invariant region (+/- 1/2) of the splice consensus sequence and is predicted to cause altered splicing leading to an abnormal or absent protein. Loss of function of the GAA gene is an established disease mechanism in autosomal recessive glycogen storage disease. The phenotype of an individual homozygous for this variant is highly specific for glycogen storage disease based on GAA enzyme activity in lymphocytes being <10% of wild type, consistent with disease (PMID: 19067231). Homozygous occurrence of this variant has been seen in three individuals with glycogen storage disease (PMID: 29122469, 24273659, 19067231), slightly supporting c.1327-2A>G as pathogenic. In summary, although additional studies are required to fully establish its clinical significance, this variant is likely pathogenic. ACMG/AMP Criteria applied: PVS1_strong, PM3_supporting, PM2, PP4 (Richards 2015).

Variant summary: GAA c.1327-2A>G is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Four predict the variant abolishes a canonical 3 prime acceptor site. The variant was absent in 250154 control chromosomes (gnomAD). c.1327-2A>G has been reported in the literature in multiple homozygous individuals affected with Glycogen Storage Disease, Type 2 (Pompe Disease) and segregated with the disease (examples: Al-Jasmi_2012 and Hamdan_2008). Hamdan_2008 demonstarted that GAA activity for the homozygous twin 1 was less than 10% of normal range (lymphocytes). These data indicate that the variant is very likely to be associated with disease. Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 and classified the variant as pathogenic (n=2, including one expert panel: ClinGen Lysosomal Storage Disorder Variant Curation Expert Panel) and likely pathogenic (n=1). Based on the evidence outlined above, the variant was classified as pathogenic.

The c.1327-2A>G variant in GAA has been reported in at least 3 homozygous and 6 compound heterozygous individuals with glycogen storage disease and segregated with the phenotype in at least one family member (Mori 2017 PMID: 29122469, Alansari 2013 PMID: 24273659, 23430803, Hamdan 2008 PMID: 19067231, Kroos 2008 PMID: 18425781). It was classified as Pathogenic on June 16, 2020 by the ClinGen Lysosomal Storage Disorder Variant Curation Expert Panel (Variation ID 972762). It has also been identified in 2/41448 of African chromosomes by gnomAD (http://gnomad.broadinstitute.org). This variant occurs in the invariant region (+/- 1/2) of the splice consensus sequence and is predicted to cause altered splicing leading to an abnormal or absent protein. This variant likely results in skipping of exon 9, resulting in an in-frame deletion of 37 amino acids and lack part of the GAA catalytic barrel (Kroos 2012, PMID 22253258; Deming 2017; DOI 10.1101/212837). GAA enzyme activity in lymphocytes of individual homozygous for the variant support an impact on protein function (Hamdan 2008 PMID: 19067231). Loss of function of the GAA gene is an established disease mechanism in autosomal recessive glycogen storage disease II. In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive glycogen storage disease II. ACMG/AMP Criteria applied: PM3_Strong, PM2_Supporting, PS3_Strong, PM4.