ClinVar Genomic variation as it relates to human health

The R239C pathogenic missense variant in the GFAP gene has been reported previously in association with Alexander disease (Brenner et al., 2001; Li et al., 2002). Functional studies indicate R239C affects the stability of GFAP protein, altering the normal solubility and organization of GFAP networks, and leading to compromised glutamate transport in astrocytes (Hsia et al., 2005; Tian et al., 2010). It was not observed in approximately 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, indicating it is not a common benign variant in these populations. The R239C variant is a non-conservative amino acid substitution, which is likely to impact secondary protein structure as these residues differ in polarity, charge, size and/or other properties. Additionally, this substitution occurs at a position that is conserved across species, and other missense mutations at the same position (R239G/H/P/L) have been reported in the Human Gene Mutation Database in association with Alexander disease (Stenson et al., 2014).

The GFAP c.715C>T (p.Arg239Cys) variant is a missense variant that is well-described as a recurrent pathogenic variant in patients with infantile- and juvenile-onset Alexander disease (Srivastava et al. 2002). Across a selection of the available literature, the p.Arg239Cys variant has been identified in a heterozygous state in 14 individuals with Alexander disease, with symptom onset ranging from six months to seven years of age (Brenner et al. 2001; Rodriguez et al. 2001; Zang et al. 2013; Jany et al. 2015). When family studies were performed, the p.Arg239Cys variant was determined to have occurred de novo (Brenner et al. 2001; Rodriguez et al. 2001; Zang et al. 2013). While many patients with the p.Arg239Cys variant are described to have a severe phenotype, the reported clinical features are variable. Of the eight patients described in Brenner et al. (2001) and Rodriguez et al. (2001), three had macrocephaly (details were not provided for the other patients), three were alive with disease duration ranging from 1.5-6.5 years, and five were deceased with disease duration ranging from 3.5-10 years. In the cohort described by Jany et al. (2015), cognition ranged from normal to severe intellectual disability. Other reported variable features included failure to thrive, emesis, swallowing difficulties, dysarthria, urinary incontinence, gait deterioration, and intermittent ataxia, among others. The p.Arg239Cys variant was absent from two individuals with non-Alexander disease leukodystrophy and from 53 control individuals without neurologic disease (Brenner et al. 2001), and is not found in the Genome Aggregation Database despite good sequencing coverage, so the variant is presumed to be rare. The p.Arg239Cys variant has been shown to affect GFAP solubility and the organization of GFAP networks (Hsiao et al. 2005). Western blotting and whole-cell patch clamp recordings have also suggested that glutamate uptake is reduced in variant-expressing astrocytes (Tian et al. 2010). This effect may play an important role in the pathogenesis of neuronal and oligodendrocyte injury and death in Alexander disease, as neurons co-cultured with astrocytes expressing the variant protein exhibited higher rates of death following glutamate challenge. Based on the collective evidence, the p.Arg239Cys variant is classified as pathogenic for Alexander disease.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0103 - Dominant negative and gain of function are suggested as mechanisms of disease in this gene and are associated with Alexander disease (MIM#203450). Functional studies have demonstrated both dominant negative and gain of function are possible mechanisms of disease, however, the latter is the most widely accepted mechanism (OMIM, GeneReviews, PMIDs: 11138011, 30355500, 31484723). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0115 - Variants in this gene are known to have variable expressivity (GeneReviews). (I) 0200 - Variant is predicted to result in a missense amino acid change from arginine to cysteine. (I) 0251 - This variant is heterozygous. (I) 0301 - Variant is absent from gnomAD (both v2 and v3). (SP) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0602 - Variant is located in a hotspot region or cluster of pathogenic variants (DECIPHER, PMID: 19386454). (SP) 0801 - This variant has very strong previous evidence of pathogenicity in unrelated individuals with infantile and juvenile Alexander disease and is considered a recurrent variant (ClinVar, PMID: 19386454). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

Variant summary: GFAP c.715C>T (p.Arg239Cys) results in a non-conservative amino acid change located in the Intermediate filament, rod domain (IPR039008) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251450 control chromosomes. c.715C>T has been reported in the literature in multiple individuals affected with Alexander Disease (examples: Brenner_2001, Rodriguez_2001). These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence showing the variant is associated with impaired autophagy leading to abnormal GFAP protein accumulation in Alexander disease (Tang_2008). The following publications have been ascertained in the context of this evaluation (PMID: 11138011, 11567214, 18276609). Five clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This recurrent pathogenic variant has been previously reported in multiple individuals with Alexander disease (PMID: 20301351, 26478912, 23364391, 11567214, 11138011, 11587071, 15732098, 34146839). Experimental evidence suggests that the c.715C>T (p.Arg239Cys) variant affects the stability of the GFAP protein and compromises glutamate transport in astrocytes (PMID: 20448479, 15840648). The c.715C>T (p.Arg239Cys) variant is absent from the gnomAD population database and thus is presumed to be rare. It affects a highly conserved amino acid and is predicted by multiple in silico tools to have a deleterious effect on protein function. Based on the available evidence, the c.715C>T (p.Arg239Cys) variant is classified as Pathogenic.

The GFAP c.715C>T (p.Arg239Cys) variant has been reported previously in association with Alexander disease (Li et al., 2002). Functional studies indicate R239C affects the stability of GFAP protein, altering the normal solubility and organization of GFAP networks, and leading to compromised glutamate transport in astrocytes (Hsia et al., 2005). The p.Arg239Cys variant is novel (not in any individuals) in gnomAD Exomes and 1000 Genomes. This variant has been submitted to ClinVar as Pathogenic. The amino acid Arg at position 239 is changed to a Cys changing protein sequence and it might alter its composition and physico-chemical properties. The amino acid change p.Arg239Cys in GFAP is predicted as conserved by GERP++ and PhyloP across 100 vertebrates. For these reasons, this variant has been classified as Pathogenic.

The GFAP c.715C>T variant is predicted to result in the amino acid substitution p.Arg239Cys. The variant is considered as the most common pathogenic variant for Alexander Disease, and in vitro functional studies suggest that the p.Arg239Cys variant may impair the filament organization, decrease solubility of GFAP, and affect glutamate transport (Hsiao et al. 2005. PubMed: 15840648; Tian et al. 2010. PubMed: 20448479). Of note, several other missense variants, affecting the same amino acid residue (p.Arg239Gly, p.Arg239His, p.Arg239Pro and p.Arg239Leu) have been reported to be causative for Alexander disease (Osorio et al. 2012 PubMed: 21940697; Brenner et al. 2001. PubMed: 11138011; Meins et al. 2002. PubMed: 12368989; Lee et al. 2006. PubMed: 17043438). This variant has not been reported in a large population database (http://gnomad.broadinstitute.org), indicating this variant is rare. This variant is interpreted as pathogenic.