ClinVar Genomic variation as it relates to human health

The p.Arg301Gln variant in GLA has been reported in at least 10 individuals with Fabry disease (Sakuraba 1990, Sawada 1996, Germain 1999, Ashton-Prolla 2000, Ge rmain 2002, Mills 2005, Lee 2010, Brady 2015, Pan 2016, Lenders 2016, Ayako 2017 ) and segregated with disease in 7 affected relatives from 3 families (Brady 201 5, Pan 2016, Saito 2017). It was absent from large population studies. Functiona l assays showed decreased alpha-Gal activity, consistent with classic Fabry dise ase (Shin 2007, Brady 2015, Pan 2016, Saito 2017). In summary, this variant meet s criteria to be classified as pathogenic for Fabry disease based upon segregati on studies, absence from controls, and functional evidence.

Variant summary: GLA c.902G>A (p.Arg301Gln) results in a conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 183457 control chromosomes (gnomAD). c.902G>A has been reported in the literature in several hemizygous individuals affected with atypical forms of Fabry Disease (FD) characterized by late onset cardiac- or renal manifestations (e.g. Sakuraba_1990, Germain_2002, Brady_2015, Kim_2019), but was also described in cases with classic FD (Germain_1999, Ashton-Prolla_2000, Lee_2010). These publications also reported decreased GLA activity in patient derived samples. These data indicate that the variant is very likely to be associated with disease. In addition, the variant protein was shown to have similar enzyme kinetic parameters to the WT in vitro, indicating that the main reason for the decreased in vivo activity could be degradation, which was also supported by the responsivity to a pharmacological chaperone (1-deoxygalactonojirimycin) treatment (Lukas_2013). Four ClinVar submitters have assessed the variant since 2014: all submitters classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This sequence change replaces arginine, which is basic and polar, with glutamine, which is neutral and polar, at codon 301 of the GLA protein (p.Arg301Gln). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with classic and atypical Fabry disease (PMID: 2171331, 8738659, 11688386, 15702404, 20505683, 23378663; Invitae). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 10715). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on GLA protein function. Experimental studies have shown that this missense change affects GLA function (PMID: 9395081, 21598360, 22241068). This variant disrupts the p.Arg301 amino acid residue in GLA. Other variant(s) that disrupt this residue have been observed in individuals with GLA-related conditions (PMID: 11322659), which suggests that this may be a clinically significant amino acid residue. For these reasons, this variant has been classified as Pathogenic.

The p.R301Q variant (also known as c.902G>A) is located in coding exon 6 of the GLAgene. This alteration results from a G to A substitution at nucleotide position 902. The arginine at codon 301 is replaced by glutamine, an amino acid with some similar properties. This alteration was initially identified in a male with atypical Fabry disease diagnosed at age 52, predominantly consisting of hypertrophic cardiomyopathy (HCM) and this individual had reduced, but not absent alpha-galactosidase A activity (Sakuraba H et al.Am J Hum Genet. 1990;47:784-789). In another study assessing the effectiveness ofpharmacologic chaperone therapy (PCT) in treating Fabry disease, this mutation was identified in a small cohort of individuals with milder Fabry disease consisting of HCM and conduction defects, but absence of stroke and renal manifestation only after age 40. Enzyme activity in white blood cells of these individuals was higher than expected in classic Fabry disease but still reduced compared to normal levels (Shin SH et alBiochem. Biophys. Res. Commun.2007 Jul;359(1):168-73). Interestingly, this alteration has also been detected in a 45 year old male with renal manifestations only, who hadmoderate proteinuria based on renal histologic findings and prominent decreases in alpha-galactosidase A activity in his plasma, urine, leukocytes, and skin fibroblasts (Sawada K et al.Clin. Nephrol. 1996 May;45:289-294). Also, a transgenic mouse model suggested that the p.R301Q mutation may cause a high frequency of misfolding in the endoplasmic reticulum, resulting in the enzyme’s rapid degradation and low residual activity in mammalian cells (Ishii S.Proc Jpn Acad, Ser B, Phys Biol Sci. 2012 ;88:18-30). This variant was previously reported in dbSNP as rs104894828, however, this variant was not reported in population-based cohorts in the 1000 Genomes Project or the NHLBI Exome Sequencing Project (ESP). In the ESP, this variant was not observed in 6503 samples (13006 alleles) with coverage at this position. Based on protein sequence alignment, this amino acid position is highly conserved in available vertebrate species.In addition, this alteration is predicted to be probably damaging and deleterious by PolyPhen and SIFT in silico analyses, respectively.Based on the available evidence to date, this alteration is interpreted as a pathogenic mutation. commonly associated with aytpical Fabry disease.

Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000010715, PMID:2171331). Functional assays showed that the variant had moderate level of impact on gene/protein function (PS3_M). A different missense change at the same codon has been reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000179546,PMID:11322659,). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.951>=0.6, 3CNET: 0.983>=0.75). A missense variant is a common mechanism . It is not observed in the gnomAD v2.1.1 dataset. Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

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; Published functional studies demonstrate a damaging effect as this variant leads to reduced enzyme activity (Lukas et al., 2013); This variant is associated with the following publications: (PMID: 25382311, 23378663, 27657681, 17532296, 17555407, 21598360, 15702404, 23935525, 22241068, 24386359, 2171331, 27356758, 27834756, 28138913, 28152038, 28728877, 27773586, 30333391, 30804731, 30477121, 12428061, 10208848, 8738659, 33673806, 33204599, 10916280, 20505683)