ClinVar Genomic variation as it relates to human health

The ASPA c.854A>C (p.Glu285Ala) missense variant has been reported in two studies in which it was found in a total of 19 individuals with Canavan disease. The p.Glu285Ala variant was identified in a homozygous state in 12 individuals, in a compound heterozygous state in two individuals and in a heterozygous state in five individuals in whom the second variant was not identified (Kaul et al. 1993; Zano et al. 2013). The p.Glu285Ala variant along with a second missense variant (p.Tyr231Ter), accounts for 98% of disease-causing alleles in the Ashkenazi Jewish population and 3% of alleles in non-Ashkenazi Jewish populations (Matalon and Michals-Matalon, 1999). The p.Glu285Ala variant was absent from 84 controls and is reported at a frequency of 0.009257 in the Ashkenazi Jewish population of the Genome Aggregation Database. Functional studies in vitro have shown the enzyme activity to be 0.02% of the wild type (Zano et al. 2013). Based on the collective evidence, the p.Glu285Ala variant is classified as pathogenic for Canavan disease. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

The p.Glu285Ala variant in ASPA is one of the most frequent pathogenic variants implicated in Canavan disease and has been reported in at least 19 Ashkenazi Jew ish individuals with the disease. Most of these individuals were homozygous (Kau l 1993). This variant has also been identified 0.04% (43/120,682) of chromosomes by the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org; dbSN P rs28940279). In vitro functional studies also provide evidence that the p.Glu 285Ala variant may impact protein function, leading to <1% of wild type enzymati c activity (Zano 2013). In summary, this variant meets our criteria to be classi fied as pathogenic for Canavan disease in an autosomal recessive manner based up on its biallelic occurrence in affected individuals and functional impact.

Expression studies found that E285A is associated with 0.3% of wild-type aspartoacylase enzyme activity as well as reduced thermal and conformational stability (Zano et al., 2013); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 23233226, 25003821, 22850825, 25333069, 21228398, 30609409, 20301412, 8037206, 31589614, 33304759, 8252036)

This sequence change replaces glutamic acid, which is acidic and polar, with alanine, which is neutral and non-polar, at codon 285 of the ASPA protein (p.Glu285Ala). This variant is present in population databases (rs28940279, gnomAD 0.9%). This missense change has been observed in individual(s) with Canavan disease (PMID: 8037206, 8252036). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 2605). 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 ASPA protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects ASPA function (PMID: 25003821). For these reasons, this variant has been classified as Pathogenic.

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 Canavan disease (MIM#271900). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0200 - Variant is predicted to result in a missense amino acid change from glutamic acid to alanine. (I) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (113 heterozygotes, 0 homozygotes). (SP) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools and very highly conserved with a major amino acid change. (SP) 0604 - Variant is not located in an established domain, motif, hotspot or informative constraint region. (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This is a common mutation reported in Askenazi Jewish population and it has been reported multiple times as pathogenic (ClinVar, PMID: 34011350). (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: The ASPA c.854A>C (p.Glu285Ala) variant involves the alteration of a conserved nucleotide. Glu285 is predicted to be part of the catalytic domain of aspartoacylase with the catalytic center of aspartoacylase involves a triad of Ser, His and Glu residues. 4/4 in silico tools predict a damaging outcome for this variant . Functional studies show that patients with this variant have very low enzyme activity. This variant was found in 43/120850 control chromosomes, predominantly observed in the European (Non-Finnish) subpopulation at a frequency of 0.000633 (42/66354). These frequencies are lower than the estimated maximal expected allele frequency of a pathogenic ASPA variant (0.0079057). Furthermore, this variant is a commonly known AJ founder mutation in the literature. The variant has been identified in many compound heterozygous and homozygous patients in the literature. In addition, multiple clinical diagnostic laboratories classified this variant as pathogenic. Taken together, this variant is classified as pathogenic.

NM_000049.2(ASPA):c.854A>C(E285A) is classified as pathogenic in the context of Canavan disease. Sources cited for classification include the following: PMID 8252036, 8037206 and 22850825. Classification of NM_000049.2(ASPA):c.854A>C(E285A) 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.

The p.E285A pathogenic mutation (also known as c.854A>C), located in coding exon 6 of the ASPA gene, results from an A to C substitution at nucleotide position 854. The glutamic acid at codon 285 is replaced by alanine, an amino acid with dissimilar properties. This is the most common ASPA mutation in the Ashkenazi Jewish population. In one study of 17 Ashkenazi Jewish individuals with Canavan disease, 12 probands were homozygous for this mutation (parental carrier status confirmed in 3 cases) and 5 probands were heterozygous for this mutation (Kaul R et al. Nat. Genet., 1993 Oct;5:118-23). A study of the crystal structure of p.E285A mutant protein showed that loss of hydrogen bonding disturbs the active site architecture, leading to altered substrate binding and lower catalytic activity (Wijayasinghe YS et al. Biochemistry, 2014 Aug;53:4970-8). Based on the supporting evidence, p.E285A is interpreted as a disease-causing mutation.

The ASPA p.Glu285Ala variant is a well-known causal variant for autosomal recessive Canavan disease (CD), and is known to be associated with 84% of CD alleles in the Ashkenazi Jewish population (Matalon_1997_PMID: 10464621). This variant was identified in dbSNP (ID: rs28940279), Clinvitae, MutDB, LOVD 3.0 and ClinVar (reported as pathogenic for CD by Invitae, Emory, Partners Laboratory for Molecular Medicine, GeneDx, Counsyl and Integrated Genetics). The variant was identified in control databases in 113 of 282830 chromosomes at a frequency of 0.0004 (Genome Aggregation Database Feb 27, 2017). The variant was observed in the following populations: Ashkenazi Jewish in 96 of 10370 chromosomes (freq: 0.009257), Other in 7 of 7228 chromosomes (freq: 0.000969), African in 2 of 24964 chromosomes (freq: 0.00008) and European (non-Finnish) in 8 of 129156 chromosomes (freq: 0.000062); it was not observed in the Latino, East Asian, European (Finnish), and South Asian populations. Multiple functional studies have shown the p.Glu285Ala mutation to cause loss of ASPA enzymatic activity (Hershfield_2007_PMID: 17391648; Mendes_2017_PMID: 28101991). The p.Glu285 residue is conserved across mammals and other organisms, and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, and MutationTaster) suggest that the variant impacts the protein. In summary, based on the above information this variant meets our laboratory’s criteria to be classified as pathogenic.

NM_000049.2:c.854A>C in the ASPA gene has an allele frequency of 0.009 in Ashkenazi Jewish subpopulation in the gnomAD database. Functional studies demonstrate that c.854A>C has disturbed the active site architecture, leading to altered substrate binding and lower catalytic activity (PMID: 25003821). Elpeleg et al reported that 18 patients with canavan disease, who are homozygous for this variant (PMID: 8037206). Pathogenic computational verdict because pathogenic predictions from DANN, DEOGEN2, EIGEN, FATHMM-MKL, M-CAP, MVP, MutationAssessor, MutationTaster, PrimateAI, REVEL and SIFT. Taken together, we interprete this variant as a Pathogenic/Likely pathogenic. ACMG/AMP criteria applied: PS3; PM3; PP3; PP4.

Variant interpreted as Pathogenic and reported on 02-10-2017 by Lab or GTR ID 1006. GenomeConnect assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. GenomeConnect staff make no attempt to reinterpret the clinical significance of the variant.