ClinVar Genomic variation as it relates to human health

The NM_000152.5:c.2238G>C variant in GAA is a missense variant predicted to cause substitution of tryptophan by cysteine at amino acid 746 (p.Trp746Cys). This variant is one of the most commonly reported variants in patients with late onset Pompe disease from East Asia (PMIDs 21757382, 31076647) and has been reported in more than 30 patients with Pompe disease (PMIDs 7981676, 18458862, 21232767, 21757382, 25093132, 25526786, 27099502, 27692865, 28433475, 29095275, 29120458, 30360039, 30897595). Because the variant often occurs in cis with pseudodeficiency variants, a conservative approach was taken when assessing data for PP4 and PM3. When pseudodeficiency variants were present or not confirmed to be absent, GAA deficiency was not used to apply PP4, and allelic data was not used for PM3 unless there was convincing evidence that the patient has Pompe disease in addition to GAA deficiency and molecular results. At least 7 Chinese patients have been reported to have the variant and were confirmed to not carry either of the pseudodeficiency variants that are common in East Asian populations (c.1726G>A (p.Gly576Ser) and c.2065G>A (p.Glu689Lys))(PMID 25526786). The patients all had documented laboratory values showing deficiency of GAA activity (PP4_Moderate). In addition, at least 4 patients with the variant and one or both pseudodeficiency variants were reported to have clinical features consistent with Pompe disease and improvements on enzyme replacement therapy (PMIDs 21232767, 25093132, 30360039). Of these patients, 8 were compound heterozygous for the variant and a GAA variant classified as pathogenic by the ClinGen LSD VCEP, phase unknown, including c.444C>G (p.Tyr148Ter), phase unknown, (PMID 25093132), c.1356delC (note that nomenclature in the paper is c.1355delC), phase unknown, (ClinVar SCV SCV001443295.1), c.1935C>A (p.Asp645Glu)(three patients, one confirmed in trans)(PMIDs 21232767, 25526786), c.2662G>T (p.Glu888Ter)(2 patients, one confirmed in trans)(ClinVar SCV001371767.1 (PMIDs 21232767, 25526786), and c.241C>T (p.Gln81Ter), phase unknown (ClinVar SCV001443296.1)(PMID 25526786)(PM3_Very Strong). The highest population minor allele frequency in gnomAD is 0.00057 (European non-Finnish) which is lower than the ClinGen LSD VCEP threshold (<0.001) for PM2, meeting this criterion (PM2_Supporting). When expressed in cultured cells, this variant has been reported to reduce GAA activity, ranging from 5-29% of the wild type activity (PMIDs 7981676, 21757382, 23430493)(PS3_Supporting). The computational predictor REVEL gives a score of 0.896, which is above the threshold of 0.7, evidence that correlates with impact to GAA function (PP3). Other missense substitutions at this amino acid position reported in patients with Pompe disease include c.2236T>C (p.Trp746Arg), c.2237G>C (p.Trp746Ser), and c.2237G>T (p.Trp746Leu). The classification for p.Trp746Cys will be used in the assessment of these other variants and therefore PM5 is not met here in order to avoid circular logic. There is a ClinVar entry for this variant (Variation ID: 265160; 2 star review status) with 14 submitters classifying the variant as pathogenic and two as likely pathogenic. 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 LSD VCEP (Specifications Version 2.0): PM3_Very Strong, PP3, PP4_Moderate, PS3_Supporting, PM2_Supporting. (Classification approved by the ClinGen LSD VCEP - Oct. 19, 2021).

The p.Trp746Cys variant in GAA has been reported in at least 2 homozygous and 24 compound heterozygous Asian individuals with juvenile- or adult-onset glycogen storage disease type II (GSDII) also known as Pompe disease (Wan 2008, Chien 20 11, Yang 2011, Liu 2014, Liong 2014, Zhang 2016, Lee 2017, Park 2017), and segre gated with GSDII in 3 affected relatives from 2 families (Yang 2011, Liu 2014). This variant has been identified in 72/126398 European and 6/18864 East Asian ch romosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitu te.org; dbSNP rs1800312). Although this variant has been seen in the general pop ulation, its frequency is low enough to be consistent with a recessive carrier f requency. Computational prediction tools and conservation analysis suggest that the p.Trp746Cys variant may impact the protein. In vitro functional studies prov ide some evidence that the p.Trp746Cys variant may impact protein function (Huie 1994, Huie 1998, Yang 2011, Nino 2013). In summary, this variant meets criteria to be classified as pathogenic for GSDII in an autosomal recessive manner. ACMG /AMP Criteria applied: PM3_VeryStrong, PS3_Moderate, PP1, PP3, PP4.

The GAA c.2238G>C; p.Trp746Cys variant (rs1800312; ClinVar Variation ID: 265160) has been identified in several individuals, both as a homozygote and in trans other pathogenic GAA variants, included in cohorts of late stage/juvenile onset Pompe disease (Yang 2011, Liu 2014, Wan 2008, Lee 2017, Liu 2018, Zhao 2019), and is the most commonly identified variant in Chinese and Taiwanese Pompe patients. Functional assessment of the p.Trp746Cys variant has revealed this variant significantly reduces GAA enzyme active compared to wild-type, although some residual activity remains (Yang 2011 and Nino 2013). Additionally, four other amino acid substitutions at codon 746 have been associated with Pompe disease: p.Trp746Arg (Nino 2013), p.Trp746Leu (Wittmann 2012), p.Trp746Gly (Labrousse 2010), and p.Trp746Ser (Kroos 2008). Functional characterization of these changes demonstrated that all have a varied impact on GAA enzyme function; ranging from less severe (p.Trp746Arg and p.Trp746Gly), to probably non-pathogenic (p.Trp746Ser) (Nino 2013). Thus, the p.Trp746Cys variant satisfies our criteria for classification as pathogenic. References: Kroos et al. Update of the Pompe disease mutation database with 107 sequence variants and a format for severity rating. Hum Mutat. 2008; 29(6): E13-26. Labrousse et al. Genetic heterozygosity and pseudodeficiency in the Pompe disease newborn screening pilot program. Mol Genet Metab. 2010; 99(4): 379-383. Lee JH et al. Targeted population screening of late onset Pompe disease in unspecified myopathy patients for Korean population. Neuromuscul Disord. 2017 Jun;27(6):550-556 Liu et al. Clinical and GAA gene mutation analysis in mainland Chinese patients with late-onset Pompe disease: identifying c.2238G > C as the most common mutation. BMC Med Genet. 2014; 15:141. Liu HX et al. Identification of Seven Novel Mutations in the Acid Alpha-glucosidase Gene in Five Chinese Patients with Late-onset Pompe Disease. Chin Med J (Engl). 2018 Feb 20;131(4):448-453. Nino et al. Identification and Functional Characterization of GAA Mutations in Colombian Patients Affected by Pompe Disease. JIMD Rep. 2013; 7: 39-48. Wan et al. Identification of eight novel mutations of the acid alpha-glucosidase gene causing the infantile or juvenile form of glycogen storage disease type II. J Neurol. 2008; 255(6): 831-838. Wittmann et al. Newborn screening for lysosomal storage disorders in hungary. JIMD Rep. 2012; 6: 117-125. Yang et al. Rapid progressive course of later-onset Pompe disease in Chinese patients. Mol Genet Metab. 2011; 104(3): 284-288. Zhao et al. Characteristics of Pompe disease in China: a report from the Pompe registry. Orphanet J Rare Dis. 2019 Apr 3;14(1):78.

Published functional studies demonstrate a damaging effect (Nio MY, 2013); In silico analysis, which includes protein predictors and evolutionary conservation, supports a deleterious effect; This variant is associated with the following publications: (PMID: 24444888, 9535769, 21232767, 21757382, 18458862, 23430493, 25526786, 28433475, 27363342, 29451150, 30943998, 31953985, 31980526, 30275481, 31589614)

The p.Trp746Cys variant in GAA has been reported in at least 35 individuals (including 26 in China, 5 in Taiwan, and 1 in Malaysia) with Glycogen Storage Disease II (PMID: 21757382, 25526786, 18458862, 7981676, 21232767, 25093132, 27099502, 29120458, 29095275, 28433475, 27099502), and has also been reported pathogenic by 10 submitters and likely pathogenic by 1 submitter in ClinVar (Variation ID: 265160). This variant has been identified in 0.057% (73/128684) of European (non-Finnish) chromosomes and 0.035% (7/19948) of East Asian chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs1800312). 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 with transfected cells provide some evidence that the p.Trp746Cys variant may impact GAA activity protein levels (PMID: 21757382, 23430493, 9535769). 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 the homozygous state and in combination with reported pathogenic and likely pathogenic variants, and in individuals with Glycogen Storage Disease II increases the likelihood that the p.Trp746Cys variant is pathogenic (PMID: 25093132, 25526786, 18458862). Four additional variants at the same position (p.Trp746Gly, p.Trp746Arg, p.Trp746Leu, and p.Trp746Ser), are pathogenic, likely pathogenic, or reported in association with disease in ClinVar (Variation ID: 556431, 499293, 284776, 188484). The phenotype of individuals heterozygous for this variant is highly specific for Glycogen Storage Disease II based on reduced GAA activity in relevant tissue, consistent with disease (PMID: 25526786, 18458862, 21757382, 21232767). In summary, this variant meets criteria to be classified as pathogenic for Glycogen Storage Disease II in an autosomal recessive manner based on multiple occurrences with pathogenic and likely pathogenic variants in individuals with Glycogen Storage Disease II and in vitro functional studies with transfected cells. ACMG/AMP Criteria applied: PM3_VeryStrong, PS3_Moderate, PM2, PP3, PP4 (Richards 2015).

PS3_Moderate, PM3_Very Strong, PM5_Supporting, PP3

This variant is interpreted as a Likely Pathogenic, for Glycogen storage disease 2, in Autosomal Recessive manner. The following ACMG Tag(s) were applied: PP3 => Multiple lines of computational evidence support a deleterious effect on the gene or gene product. PM2 => Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium. PS3 => Well-established functional studies show a deleterious effect (PMID:18458862).

The GAA c.2238G>C (p.Trp746Cys) variant has been reported in at least six studies in which it is found in a total of 31 individuals with glycogen storage disease, type II, including three in a homozygous state, 27 in a compound heterozygous state, and one in a heterozygous state in whom a second variant was not identified (Huie et al. 1994; Wan et al. 2008; Yang et al. 2011; Liu et al. 2014; Liong et al. 2014; Zhang et al. 2016). Ten of the compound heterozygous individuals were found to carry a second GAA variant in cis with the p.Trp746Cys variant (Huie et al. 1994; Yang et al. 2011). However, functional studies in cell lines demonstrated that expression of the p.Trp746Cys variant alone resulted in enzymatic activity that was 10-29% of wild type GAA activity (Huie et al. 1994; Huie et al. 1998; Yang et al. 2011; Nino et al. 2013). The p.Trp746Cys variant was absent from 100 controls and is reported at a frequency of 0.00048 in the European (non-Finnish) population of the Exome Aggregation Consortium. Based on the evidence, the p.Trp746Cys 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.

NM_000152.3(GAA):c.2238G>C(W746C) is classified as pathogenic in the context of Pompe disease. Sources cited for classification include the following: PMID 25093132, 9535769, 23430493, 25526786, 21757382 and 27099502. Classification of NM_000152.3(GAA):c.2238G>C(W746C) 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.‚Äã

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as pathogenic. Following criteria are met: 0102 - Loss-of-function is a known mechanism of disease for this gene and is associated with glycogen storage disease II (MIM#232300). (N) 0106 - This gene is known to be associated with autosomal recessive disease. (I) 0200 - Variant is predicted to result in a missense amino acid change from tryptophan to cysteine. (I) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD <0.01 for a recessive condition (v2: 87 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v2) (22 heterozygotes, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools and highly conserved with a major amino acid change. (P) 0600 - Variant is located in an annotated glycosyl hydrolases family 31 domain (Decipher). (I) 0801 - This variant has very strong previous evidence of pathogenicity in unrelated individuals. This variant is classified as likely pathogenic or pathogenic in Clinvar and has been reported as homozygous or compound heterozygous in individuals with Pompe disease or glycogen storage disease II (ClinVar; PMIDs: 18458862, 25526786, 31931849, 32126021). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

This sequence change replaces tryptophan, which is neutral and slightly polar, with cysteine, which is neutral and slightly polar, at codon 746 of the GAA protein (p.Trp746Cys). This variant is present in population databases (rs1800312, gnomAD 0.06%). This missense change has been observed in individual(s) with Pompe disease (PMID: 18458862, 21232767, 21757382, 23430493, 25093132, 25526786, 27099502). 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: 265160). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae 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: 7981676, 9535769, 21757382). For these reasons, this variant has been classified as Pathogenic.

The p.W746C pathogenic mutation (also known as c.2238G>C), located in coding exon 15 of the GAA gene, results from a G to C substitution at nucleotide position 2238. The tryptophan at codon 746 is replaced by cysteine, an amino acid with highly dissimilar properties. This alteration has been reported in the compound heterozygous and homozygous states in individuals with Pompe disease, mainly late onset disease (Wan L et al. J Neurol, 2008 Jun;255:831-8; Chien YH et al. J Pediatr, 2011 Jun;158:1023-1027.e1; Yang CC et al. Mol Genet Metab, 2011 Nov;104:284-8; Fu Liong H et al. Case Rep Neurol Med, 2014 Jun;2014:926510; Liu X et al. BMC Med Genet, 2014 Dec;15:141; Chu YP et al. Neuromuscul Disord, 2016 Dec;26:873-879; Zhang B et al. Neuropsychiatr Dis Treat, 2016 Mar;12:713-7; Lee JH et al. Neuromuscul Disord, 2017 Jun;27:550-556; Park HJ et al. Clin Genet, 2017 Mar;91:403-410; Kim MS et al. Korean J Pediatr, 2019 Jun;62:224-234; Ficicioglu C et al. Int J Neonatal Screen, 2020 Nov;6:[ePub ahead of print]; Jia X et al. Aging (Albany NY), 2020 Mar;12:4268-4282; Sudri&eacute;-Arnaud B et al. Diagnostics (Basel), 2021 Feb;11:[ePub ahead of print]; Zhao HH et al. Ann Transl Med, 2021 Dec;9:1803; Si X et al. CNS Neurosci Ther, 2022 Oct;28:1651-1654; Zhang H et al. Front Neurol, 2022 Mar;13:839263). This alteration has also been shown to reduce enzyme activity in both patient-derived cells and transfected cells (Ni&ntilde;o MY et al. JIMD Rep, 2013 Apr;7:39-48; Liu HX et al. Chin Med J (Engl), 2018 Feb;131:448-453). In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

GAA: PM3:Very Strong, PM1, PM2, PP4:Moderate, PS3:Supporting

The GAA c.2238G>C variant is predicted to result in the amino acid substitution p.Trp746Cys. This variant has been reported, in the homozygous or compound heterozygous state, in numerous glycogen storage disease type II (GSD II) patients. In most cases, it has been associated with late-onset/juvenile-onset GSD II (e.g., Wan et al. 2008. PubMed ID: 18458862; Chien et al. 2011. PubMed ID: 21232767; Liu et al. 2018. PubMed ID: 29451150). It has been reported to be the most common late-onset Pompe disease (LOPD) variant among the mainland Chinese population (Liu et al. 2014. PubMed ID: 25526786). In functional studies, the p.Trp746Cys substitution has been reported to reduce GAA enzyme activity to ~5-10% of wild-type, and is typically considered a mild variant based on this level of residual enzyme activity (Huie et al. 1998. PubMed ID: 9535769; Yang et al. 2011. PubMed ID: 21757382; Niño et al. 2013. PubMed ID: 23430493). This variant is reported in 0.057% of alleles in individuals of European (Non-Finnish) descent in gnomAD. Different substitutions of the same amino acid (p.Trp746Arg, p.Trp746Gly, p.Trp746Ser) have also been reported in association with GSD II (Human Gene Mutation Database). The c.2238G>C (p.Trp746Cys) variant is interpreted as pathogenic.