ClinVar Genomic variation as it relates to human health

The p.Arg302Gln variant in PRKAG2 has been reported in numerous individuals with variable clinical features (including cardiac hypertrophy, Wolff-Parkinson-White syndrome (WPW) and other conduction system abnormalities) and segregated with disease in greater than 100 individuals from at least 13 families (Gollob 2001 PMID: 11407343; Arad 2002 PMID: 11827995; Sternick 2006 PMID: 16836667; Charron 2007 PMID: 17483151; Tan 2008 PMID: 19808419; Alfares 2015 PMID: 25611685; Walsh 2017 PMID: 27532257; Pu 2015 PMID: 25997934; Zhan 2018 PMID: 29452156; Jaaskelainen 2019 PMID: 30775854; Robyns 2020 PMID: 31513939; Hu 2020 PMID: 32681253). This variant has also been reported by other clinical laboratories in ClinVar (Variation ID: 6846). It was absent from large population studies. In vitro and in vivo functional studies, including animal models in mouse, have demonstrated that this variant impacts protein function and causes disease that is similar to what has been observed in humans (Thorn 2013 PMID: 23829931; Zhang 2013 PMID: 23778007; Yavari 2016 PMID: 27133129; Zhan 2018 PMID: 29452156). Computational prediction tools and conservation analyses do not provide strong support for or against an impact to the protein. In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant PRKAG2-associated cardiac disease. ACMG/AMP Criteria applied: PS4, PP1_Strong, PM2, PS3_Moderate.

Variant summary: PRKAG2 c.905G>A (p.Arg302Gln) results in a conservative amino acid change in the encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251376 control chromosomes. c.905G>A has been widely reported in the literature in multiple individuals affected with Hypertrophic Cardiomyopathy With Wolff-Parkinson-White (example, Gollob_2001, Sternick_2006, Arad_2002, Tan_2008). These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function (example, Scott_2004). The most pronounced variant effect results in impaired binding of AMP to PRKAG2 protein. Multiple clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 with complete consensus as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

Reported in families with conduction disease and little to no history of LVH or WPW (Sternick et al., 2006; Charron et al., 2007), demonstrating phenotypic variability; Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Functional studies have demonstrated that transgenic animal models harboring p.(R302Q) develop a phenotype congruent to the human disease spectrum, including reduced cardiac function, LVH, prolonged QRS, ventricular pre-excitation, increased cardiac glycogen stores, and/or reduced AMPK activity (Sidhu et al., 2005; Folmes et al., 2009; Thorn et al., 2013; Zhang et al., 2014); Located within a CBS domain; these domains regulate binding of PRKAG2 to ATP, ADP, and AMP; This variant is associated with the following publications: (PMID: 23829931, 19920047, 26729852, 11827995, 34656342, 30847666, 23992123, 27189955, 27864312, 25611685, 26496977, 25997934, 27532257, 27133129, 27496753, 23778007, 17483151, 23810891, 11407343, 24497343, 15611370, 20031621, 16836667, 29298659, 26333379, 31513939, 30775854, 31737537, 32150461, 33662488, 32681253, 33906374, 33244021, 33673806, 32646569, 14722619, 26582918)

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 in this gene and is associated with hypertrophic cardiomyopathy 6 (MIM#600858) and Wolff-Parkinson-White syndrome (MIM#194200). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0115 - Variants in this gene are known to have variable expressivity between families and also within the same family (PMID: 11407343, 28431061). (I) 0200 - Variant is predicted to result in a missense amino acid change from arginine to glutamine. (I) 0251 - This variant is heterozygous. (I) 0302 - Variant is present in gnomAD (v3) <0.001 for a dominant condition (1 heterozygote, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v2) (1 heterozygote, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0600 - Variant is located in the annotated CBS1 domain (Uniprot, PDB). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been previously reported as pathogenic in multiple individuals (ClinVar). This variant has also been shown to segregate with hypertrophy and Wolff-Parkinson-White syndrome in many affected individuals across several families (PMID: 11407343, 28431061). (SP) 1002 - This variant has moderate functional evidence supporting abnormal protein function. Functional studies have shown that AMPK enzymatic activity is significantly reduced by this variant and transgenic mouse models carrying this variant recapitulate the human phenotype (PMID: 23778007, 15611370). (SP) 1205 - This variant has been shown to be maternally inherited. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

This variant is interpreted as a Pathogenic, for Cardiomyopathy, familial hypertrophic 6, in Autosomal Dominant manner. The following ACMG Tag(s) were applied: PM2 => Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium. PS3-Very Strong => PS3 upgraded in strength to Very Strong (PMID:14722619,11827995,15611370,28917552). PP1-Moderate => PP1 upgraded in strength to Moderate (PMID:11407343).

This variant was classified as: Pathogenic. The following ACMG criteria were applied in classifying this variant: PS1,PS3,PM2,PP3.

The variant was co-segregated with Wolff-Parkinson-White syndrome in multiple affected family members with additional meioses (PMID: 25997934, 16836667) (PP1_S). The variant has been observed in multiple (>3) similarly affected unrelated individuals(PMID: 25997934, 16836667, PS4_S). It has been previously reported as de novo in a similarly affected individual (PMID: 28431061, PS2_S). Functional studies provide strong evidence of the variant having a damaging effect on the gene or gene product (PMID: 14722619, 15611370, 20031621, 23992123, PS3_S). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.838, 3CNET: 0.991, PP3_P). A missense variant is a common mechanism associated with Wolff-Parkinson-White syndrome (PP2_P). It is not observed in the gnomAD v2.1.1 dataset (PM2_M). A different missense change at the same codon has been reported to be associated with PRKAG2 related disorder (PMID:32259713, PM5_P). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

The p.R302Q pathogenic mutation (also known as c.905G>A), located in coding exon 7 of the PRKAG2 gene, results from a G to A substitution at nucleotide position 905. The arginine at codon 302 is replaced by glutamine, an amino acid with highly similar properties. This mutation has been reported in multiple individuals in association with Wolff-Parkinson-White syndrome, cardiac conduction system disease, and hypertrophic cardiomyopathy, or a combination of these presentations, and has been seen to segregate with disease in many individuals from multiple unrelated families (Gollob et al. N Engl J Med. 2001 Jun;344(24):1823-31; Arad et al. J Clin Invest. 2002 Feb;109(3):357-62; Sternick et al. J Cardiovasc Electrophysiol. 2006 Jul; 17(7):724-32; Charron et al. Europace. 2007 Aug;9:597-600; Tan et al. Circ Arrhythm Electrophysiol. 2008 Oct;1:276-81; Hathaway J et al. BMC Cardiovasc Disord, 2021 03;21:126). At least one de novo case has also been reported (Thevenon J et al. Europace, 2017 Apr;19:651-659). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). In addition, in vitro functional studies and in vivo studies of transgenic mice have demonstrated abnormal protein function and increased cardiac glycogen storage as a result of this mutation (Scott et al. J Clin Invest. 2004 Jan;113:274-84; Sidhu et al. Circulation. 2005 Jan;111:21-9; Folmes et al. Circ Cardiovasc Genet. 2009 Oct;2:457-66; Zhang et al. J Cardiol. 2013 Oct;62:241-8; Thorn et al. EJNMMI Res. 2013;3:48). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

This sequence change replaces arginine, which is basic and polar, with glutamine, which is neutral and polar, at codon 302 of the PRKAG2 protein (p.Arg302Gln). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with hypertrophy and Wolff-Parkinson-White syndrome (PMID: 16836667, 25997934). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 6846). 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 PRKAG2 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects PRKAG2 function (PMID: 14722619, 15611370, 20031621, 23992123). For these reasons, this variant has been classified as Pathogenic.

The patient had genetic testing with the GeneDx laboratory with their comprehensive cardiomyopathy panel. The test included 77 genes associated with various hereditary cardiomyopathies: ABCC9, ACTC (ACTC1), ACTN2, ANKRD1, BAG3, BRAF, CAV3, CRYAB, CSRP3, DES, DMD, DSC2, DSG2, DSP, DTNA, EMD, FKTN, GATAD1, GLA, HRAS, ILK, JPH2, JUP, KRAS, LAMA4, LAMP2, LDB3 (ZASP), LMNA, MAP2K1, MAP2K2, MTND1, MTND5, MTND6, MTTD, MTTG, MTTH, MTTI, MTTK, MTTL1, MTTL2, MTTM, MTTQ, MTTS1, MTTS2, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, MYPN, NEBL, NEXN, NRAS, PDLIM3, PKP2, PLN, PRKAG2, PTPN11, RAF1, RBM20, RYR2, SCN5A, SGCD, SOS1, TAZ, TCAP, TMEM43,TMPO, TNNC1, TNNI3, TNNT2, TPM1, TTN, TTR, VCL. Analysis included both sequencing and array-based comparative genome hybridization to look for duplications and deletions (excluding mitochondrial genes, FKTN, GATAD1) Results showed that a variant was found (see report below): -p.Arg302Gln (c.905G>A) in the PRKAG2 gene. This variant is reviewed in detail below. Other cases of p.Arg302Gln (R302Q): -Gollob M et al., 2001; found R302Q to co-segregate with WPW syndrome in two unrelated families, while it was not observed in 300 control alleles. High penetrance with variable expressivity of WPW (pre excitation). Initial presentations included palpitations, presyncope and syncope with first onset in late adolescence or the third decade of life. Paroxsysmal a-fib or flutter occered in assocation 40% of the time. Arad M et al., 2002; reported R302Q in four separate families with a combination of WPW, conduction system disease, and cardiac hypertrophy. PRKAG2 mutations Arg302Gln, Thr400Asn, and Asn488Ile include myocyte enlargement and minimal interstitial fibrosis, these mutations were not associated with myocyte and myofibrillar disarray, the pathognomonic features of hypertrophic cardiomyopathy caused by sarcomere protein mutations. -Tan et al., 2008 reported a family of 17 individuals of a 5 generation family. Cardiac hypertrophy was found in 7 / 10 mutation carriers and ECGs were compatible with pre-excitation in 13 subjects with AV block in 5 subjects. All mutation carriers had electrocardiographic evidence of preexcitation, AV block, or both. Three individuals had high-grade AV block with preexcited conducted beats. Electrophysiological studies in 3 individuals revealed bypasses with AV node–like properties. Histopathologic studies of 1 suddenly deceased mutation carrier revealed concentric hypertrophy of the left ventricle with extensive myocardial disarray associated with slight interstitial fibrosis but no lysosomal-bound glycogen. Moreover, there were 3 small nodoventricular tracts (Mahaim fibers) passing through the central fibrous body and connecting the AV node with the working myocardium of the interventricular septum -Zhang L et al., 2010; R302Q to cosegregate with WPW syndrome in one family with five affected individuals with significant atrial fibrillation and no hypertrophy. -Tian Pu et al., 2015 reported a proband with myocardial hypertrophy, pre-excitation with a short PR interval on ECG in a Chinese individual. -This PRKAG2 mutation also has been observed in other unrelated individuals tested for HCM at GeneDx (pathogenic), LMM (11 individuals in clinvar - pathogenic), Blueprint Genetics (2 individuals in clinvar - pathogenic). Functional Studies: -Folmes K et al., 2009: expression of R302Q PRKAG2 in transgenic mice was shown to alter protein function and leading to increased glycogen content of the heart tissue, explaining the glycogen storage cardiomyopathy caused by PRKAG2 mutations In Silico: -Change from large size and basic (R) to medium size and polar (Q). -Polyphen 2 predicts this change to be possibly damaging with a HumVar score of 0.447. Population Studies and controls: -R302Q was absent in 300 control alleles in Gollob M et al. -NHLBI Exome sequencing project reports R302Q was not observed in approximately 6,000 individuals of European and African American backgrounds -R302Q is absent in the exac.broadinstitute.org database which includes 60,000+ individuals who were not selected for cardiovascular disease, but R302L was seen in one individual out of 60,000+ individuals meaning it's sufficiently rare/absent in the general population.