ClinVar Genomic variation as it relates to human health

The p.Glu427Gln (commonly referred to in the literature as p.Glu399Gln) is missense variant in the ALDH7A1 gene. This variant is well reported in the literature, and had been reported to be the most prevalent pathogenic variant in ALDH7A1 (PMID: 20301659). The variant has been reported by multiple clinical laboratories as pathogenic in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/RCV000019610.29/). Functional studies have shown that the p.Glu427Gln results in an absence of detectable enzyme activity in a CHO in vitro cell model (PMID: 16491085). The highest reported allele frequency in the population databases is 0.0006% (81/126556 alleles). In silico modeling predicts this variant as damaging and it is highly conserved. Based on the available evidence, the p.Glu427Gln variant is classified as pathogenic.

The homozygous p.Glu427Gln variant in ALDH7A1 was identified by our study in 1 individual with pyridoxine-dependent epilepsy. The variant has been reported in at least 10 individuals of European and unknown ethnicity with pyridoxine-dependent epilepsy (PMID: 16491085, 19128417), and has been identified in 0.06% (79/129032) of European non-Finnish, 0.02% (4/19918) of East Asian, and 0.01% (4/35418) of Latino chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs121912707). Although this variant has been seen in the general population, its frequency is not high enough to rule out a pathogenic role. This variant has also been reported in ClinVar (Variation ID: 17994) as pathogenic by multiple submitters and as likely pathogenic by UCLA Clinical Genomics Center. In vitro functional studies provide some evidence that the p.Glu427Gln variant may impact protein function (PMID: 22784480, 16491085). 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 at least 5 affected homozygotes, and in combination with 2 reported pathogenic variants, and in at least 10 individuals with pyridoxine-dependent epilepsy increases the likelihood that the p.Glu427Gln variant is pathogenic (VariationID: 17995, 204852; PMID: 16491085, 19128417). The p.Glu427Gln variant is located in a region of ALDH7A1 that is essential to protein folding and stability, suggesting that this variant is in a mutational hotspot and slightly supports pathogenicity (PMID: 16491085, 31652343). One additional likely pathogenic variants, resulting in a different amino acid change at the same position (p.Glu427Gly), have been reported in association with disease in the literature, supporting that a change at this position may not be tolerated (PMID: 19128417). In summary, the p.Glu427Gln variant is pathogenic based off of our findings of multiple reports in ClinVar, and the literature. ACMG/AMP Criteria applied: PM3_strong, PM5_supporting, PM1, PS3_moderate, PP3 (Richards 2015).

Published functional studies demonstrate the variant abolishes enzyme activity (Mills et al., 2006; Coulter-Mackie et al., 2012); 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: 31652343, 16491085, 26555630, 17068770, 17088338, 17721876, 26224730, 29056246, 31164858, 31980526, 20370816, 22371912, 22784480, 27438048, 26995068, 26943461, 16159904, 28832562, 29453417, 30609409, 29401530, 31737911, 31564432, 23430810, 30043187, 31130284, 23022070, 31589614, 34426522, 32685344)

The ALDH7A1 c.1279G>C; p.Glu427Gln variant (rs121912707), also known as Glu399Gln for legacy nomenclature, is reported as a common pathogenic variant that has been identified both homozygous and compound heterozygous in several individuals with autosomal recessive epilepsy (Bennett 2009, Mefford 2015, Monies 2019, Schmitt 2010). Additionally, other variants at this codon (p.Glu427Asp, p.Glu427Gly) have also been reported in affected individuals (Bennett 2009). The p.Glu427Gln variant is reported in ClinVar (Variation ID: 17994). It is observed in the general population with an overall allele frequency of 0.03% (93/282634 alleles) in the Genome Aggregation Database (v2.1.1). Computational analyses predict that this variant is deleterious (REVEL: 0.944). In support of these predictions, functional analyses show an effect of this variant on enzyme activity (Coulter-Mackie 2012). Based on available information, this variant is considered to be pathogenic. References: Bennett CL et al. Prevalence of ALDH7A1 mutations in 18 North American pyridoxine-dependent seizure (PDS) patients. Epilepsia. 2009 May;50(5):1167-75. PMID: 19128417. Coulter-Mackie MB et al. Overexpression of human antiquitin in E. coli: enzymatic characterization of twelve ALDH7A1 missense mutations associated with pyridoxine-dependent epilepsy. Mol Genet Metab. 2012 Aug;106(4):478-81. PMID: 22784480. Mefford HC et al. Intragenic deletions of ALDH7A1 in pyridoxine-dependent epilepsy caused by Alu-Alu recombination. Neurology. 2015 Sep 1;85(9):756-62. PMID: 26224730. Monies D et al. Lessons Learned from Large-Scale, First-Tier Clinical Exome Sequencing in a Highly Consanguineous Population. Am J Hum Genet. 2019 Jun 6;104(6):1182-1201. PMID: 31130284. Schmitt B et al. Seizures and paroxysmal events: symptoms pointing to the diagnosis of pyridoxine-dependent epilepsy and pyridoxine phosphate oxidase deficiency. Dev Med Child Neurol. 2010 Jul;52(7):e133-42. PMID: 20370816.

The p.Glu427Gln variant in ALDH7A1 (also referred to as p.Glu399Gln in the literature) has been reported in at least 3 homozygous and 7 compound heterozygous individuals with pyridoxine-dependent epilepsy (Mills 2006 PMID: 16491085, Schmitt 2010 PMID: 20370816, Nam 2012 PMID: 22371912, Proudfoot 2012 PMID: 23430810, van Karnebeek 201 PMID: 230220702). This variant has also been identified in 45/66588 of European chromosomes by the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org; dbSNP rs121912707). Although this variant has been seen in the general population, its frequency is low enough to be consistent with a recessive carrier frequency. In addition, in vitro functional studies provide evidence that the p.Glu427Gln variant impacts protein function (Mills 2006 PMID: 16491085, Coulter-Mackie 2012 PMID: 22784480). In summary, this variant meets our criteria to be classified as pathogenic for pyridoxine-dependent epilepsy in an autosomal recessive manner based upon its co-occurrence with pathogenic variants in affected individuals and functional evidence. ACMG/AMP criteria applied: PM3_VeryStrong, PS3, PP3.

Across a selection of available literature, the ALDH7A1 c.1279G>C (p.Glu427Gln) missense variant, also referred to as p.Glu399Gln, has been reported in at least seven studies and identified in a total of 15 individuals, including in six in a homozygous state and in nine in a compound heterozygous state, all of whom have a diagnosis of pyridoxine-dependent epilepsy or seizures (Mills et al. 2006; Bennett et al. 2009; Schmitt et al. 2010; Nam et al. 2012; Proudfoot et al. 2013; Tlili et al. 2013; Mefford et al. 2015). The p.Glu427Gln variant was absent from 230 controls but is reported at a frequency of 0.00081 in the European American population of the Exome Sequencing Project. Functional studies using transfected CHO cells were performed by Mills et al. (2006) and found α-AASA dehydrogenase activity to be undetectable. Coulter-Mackie et al. (2012) used transfected E. coli and observed enzyme activity levels below the level of detection for the assay. Based on the collective evidence, the p.Glu427Gln variant is classified as pathogenic for pyridoxine-dependent epilepsy. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

The p.E427Q pathogenic mutation (also known as c.1279G>C), located in coding exon 14 of the ALDH7A1 gene, results from a G to C substitution at nucleotide position 1279. The glutamic acid at codon 427 is replaced by glutamine, an amino acid with highly similar properties. This mutation has been described in numerous unrelated individuals with pyroxidine-dependent seizures in both homozygous and compound heterozygous states, and is reported to be the most common ALDH7A1 mutation, accounting for approximately 30% of mutant alleles (Mills PB et al. Nat. Med., 2006 Mar;12:307-9; Plecko B et al. Hum. Mutat., 2007 Jan;28:19-26; Bennett CL et al. Epilepsia, 2009 May;50:1167-75; Nam SH et al. Ann. Clin. Lab. Sci., 2012;42:65-72; Mefford HC et al. Neurology, 2015 Sep;85:756-62; Butler KM et al.<span style="background-color:rgb(255, 255, 255); color:rgb(51, 51, 51); font-family:sans-serif,arial,verdana,trebuchet ms; font-size:13px"> Pediatr. Neurol.<span style="background-color:rgb(255, 255, 255); color:rgb(51, 51, 51); font-family:sans-serif,arial,verdana,trebuchet ms; font-size:13px">, 2017 Dec;77:61-66<span style="background-color:rgb(255, 255, 255); color:rgb(51, 51, 51); font-family:sans-serif,arial,verdana,trebuchet ms; font-size:13px">). Enzyme activity in transfected CHO cells and E. coli cells have demonstrated undetectable or extremely low (<3% of WT) enzyme activity (Mills PB et al. Nat. Med., 2006 Mar;12:307-9; Coulter-Mackie MB et al. Mol. Genet. Metab., 2012 Aug;106:478-81). <span style="background-color:rgb(255, 255, 255); color:rgb(51, 51, 51); font-family:sans-serif,arial,verdana,trebuchet ms; font-size:13px">In addition, this alteration is predicted to be probably damaging, deleterious, and deleterious by PolyPhen and<span style="background-color:rgb(255, 255, 255); color:rgb(51, 51, 51); font-family:sans-serif,arial,verdana,trebuchet ms; font-size:13px"> SIFT<span style="background-color:rgb(255, 255, 255); color:rgb(51, 51, 51); font-family:sans-serif,arial,verdana,trebuchet ms; font-size:13px"> in silico<span style="background-color:rgb(255, 255, 255); color:rgb(51, 51, 51); font-family:sans-serif,arial,verdana,trebuchet ms; font-size:13px"> analyses, respectively.Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000017994, PMID:16491085, PS1_S). A different missense change at the same codon has been reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000204865,VCV000944165, PMID:19128417,19128417, PM5_M). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.944, 3CNET: 0.996, PP3_P). It is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.000329, PM2_M). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

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 pyridoxine-dependent epilepsy (MIM#266100). (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 glutamine. (I) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (93 heterozygotes, 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 NAD binding site within the aldehyde dehydrogenase domain (NCBI, DECIPHER). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported many times as pathogenic and likely pathogenic, and is described as the most common pathogenic variant among European patients (ClinVar, PMID: 30043187). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

ALDH7A1 NM_001182.4 exon 14 p.Glu427Gln (c.1279G>C): This variant is a well known pathogenic variant and has been reported in the literature in numerous individuals with pyridoxine dependent epilepsy in the homozygous and compound heterozygous state (also reported as p.Glu399Gln; selected publications: Mills 2006 PMID:16491085, Bennett 2009 PMID:19128417, Schmitt 2010 PMID:20370816, van Karnebeek 2012 PMID:23022070, Mefford 2015 PMID:26224730, Coughlin 2019 PMID:30043187). Multiple publications suggest that this variant represents 1/3rd of mutated alleles for this gene (Bennett 2009 PMID:19128417, Mefford 2015 PMID:26224730). This variant is present in 0.04% (29/68034) of European alleles in the Genome Aggregation Database (https://gnomad.broadinstitute.org/variant/5-126552059-C-G?dataset=gnomad_r3). Please note, disease causing variants may be present in control databases at low frequencies, reflective of the general population, carrier status, and/or variable expressivity. This variant is present in ClinVar, with several labs classifying this variant as Pathogenic (Variation ID:17994). Evolutionary conservation and computational predictive tools suggest that this variant may impact the protein. In addition, functional studies utilizing chinese hamster ovary cells and e.coli assays have shown a deleterious effect of this variant (Mills 2006 PMID:16491085, Coulter-Mackie 2012 PMID:22784480). Furthermore, other variants at this position (p.Glu427Gly/p.Glu399Gly and p.Glu427Asp/p.Glu399Gly) have also been reported in association with disease supporting that this codon has functional importance. In summary, this variant is classified as Pathogenic.

This sequence change replaces glutamic acid, which is acidic and polar, with glutamine, which is neutral and polar, at codon 427 of the ALDH7A1 protein (p.Glu427Gln). This variant is present in population databases (rs121912707, gnomAD 0.06%). This missense change has been observed in individual(s) with pyridoxine dependent epilepsy (PMID: 16491085, 19128417, 22371912, 26224730). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 17994). 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 ALDH7A1 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects ALDH7A1 function (PMID: 16491085). For these reasons, this variant has been classified as Pathogenic.

Criteria applied: PM3_VSTR,PM5_STR,PP3

The most common pathogenic variant, accounting for 33% of pathogenic variants