The variant is observed at an extremely low frequency in the gnomAD v4.1.0 dataset (total allele frequency: 0.006%). Predicted Consequence/Location: Missense variant. The majority of the known disease-causing variants of this gene are variants expected to result in premature termination of the protein. The variant is in trans with NM_000019.4:c.481T>C. In silico tool predictions suggest damaging effect of the variant on gene or gene product [3Cnet: 0.93 (> 0.75, sensitivity 0.96 and precision 0.92)].The same nucleotide change resulting in the same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000198030 / PMID: 17236799 / 3billion dataset). A different missense change at the same codon (p.Asn158Asp) has been reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000092297 / PMID: 7749408). The variant is in trans with the other variant. Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline.

Variant summary: ACAT1 c.473A>G (p.Asn158Ser) results in a conservative amino acid change located in the Thiolase, N-terminal of the encoded protein sequence. Three of five in-silico tools predict a benign effect of the variant on protein function. The variant allele was found at a frequency of 0.00031 in 251402 control chromosomes. This frequency is not significantly higher than expected for a pathogenic variant in ACAT1 causing Mitochondrial Acetoacetyl-CoA Thiolase Deficiency (0.00031 vs 0.0029), allowing no conclusion about variant significance. c.473A>G has been reported in the literature in individuals affected with Mitochondrial Acetoacetyl-CoA Thiolase Deficiency and has been subsequently cited by others (example, Sakurai_2007, Sarafoglou_2011, Abdelkreem_2019). These data indicate that the variant is likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function (Sakurai_2007). The most pronounced variant effect results in a loss of catalytic activity due to an inability to fold into a tetramer configuration essential for the tertiary structure. A different missense substitution (p.Asn158Asp) at this location has been reported in patients with this disorder further corroborating a critical role of the Aspargine residue for protein function. Two 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 (n=1)/likely pathogenic (n=1). Based on the evidence outlined above, the variant was classified as pathogenic.

The ACAT1 c.473A>G; p.Asn158Ser variant (rs199524907), is reported in the literature in three individuals affected with alpha-methylacetoacetic aciduria (Abdelkreem 2019, Sakurai 2007, Sarafoglou 2011). This variant is also reported in ClinVar (Variation ID: 198030). This variant is found in the general population with an allele frequency of 0.03% (81/282788 alleles) in the Genome Aggregation Database. Additionally, another amino acid substitution at this codon (c.472A>G; p.Asn158Asp) has been reported in individuals with alpha-methylacetoacetic aciduria and is considered likely pathogenic (Otsuka 2016, Sakura 2007, Wakazono 1995). Functional analysis of the variant protein show a loss of catalytic activity (Abdelkreem 2019, Sakurai 2007). Computational analyses are uncertain whether this variant is neutral or deleterious (REVEL: 0.524). Based on available information, the c.473A>G; p.Asn158Ser variant is considered to be likely pathogenic. References: Abdelkreem E et al. Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency. Hum Mutat. 2019 Oct;40(10):1641-1663. PMID: 31268215. Otsuka H et al. Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site. Mol Med Rep. 2016 Nov;14(5):4906-4910. PMID: 27748876. Sakurai S et al. Kinetic and expression analyses of seven novel mutations in mitochondrial acetoacetyl-CoA thiolase (T2): identification of a Km mutant and an analysis of the mutational sites in the structure. Mol Genet Metab. 2007 Apr;90(4):370-8. PMID: 17236799. Sarafoglou K et al. Siblings with mitochondrial acetoacetyl-CoA thiolase deficiency not identified by newborn screening. Pediatrics. 2011 Jul;128(1):e246-50. PMID: 21669895. Wakazono et al. Molecular, biochemical, and clinical characterization of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in two further patients. Hum Mutat. 1995;5(1):34-42. PMID: 7728148.

The c.473A>G (p.N158S) alteration is located in exon 6 (coding exon 6) of the ACAT1 gene. This alteration results from a A to G substitution at nucleotide position 473, causing the asparagine (N) at amino acid position 158 to be replaced by a serine (S). Based on data from the Genome Aggregation Database (gnomAD) database, the ACAT1 c.473A>G alteration was observed in 0.03% (81/282788) of total alleles studied, with a frequency of 0.22% (78/35438) in the Latino subpopulation. This alteration has been reported in the compound heterozygous state with another alteration in patients with clinical and biochemical features of alpha-methylacetoacetic aciduria (Sakurai, 2007; Sarafoglou, 2011). In addition, another pathogenic mutation (p.N158D) has been reported affecting the same amino acid position (Sakurai, 2007). Based on transient expression analysis of mutant cDNA, the N158S alteration demonstrated protein instability in a temperature-sensitive manner and no residual enzyme activity (Sakurai, 2007). Based on the available evidence, this alteration is classified as pathogenic.

Published functional studies demonstrate a damaging effect: temperature sensitive instability, with faint to no detection of mutant protein at different temperatures as compared to wild-type protein (PMID: 17236799); 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: 31268215, 21669895, 17236799, 39519275)

This sequence change replaces asparagine, which is neutral and polar, with serine, which is neutral and polar, at codon 158 of the ACAT1 protein (p.Asn158Ser). This variant is present in population databases (rs199524907, gnomAD 0.2%). This missense change has been observed in individual(s) with acetoacetyl-CoA-thiolase deficiency (PMID: 17236799, 21669895). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 198030). Invitae Evidence 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 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 ACAT1 protein function. Experimental studies have shown that this missense change affects ACAT1 function (PMID: 17236799, 21669895). This variant disrupts the p.Asn158 amino acid residue in ACAT1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 7749408, 17236799, 27748876). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

Missense variation is an established mechanism of disease for beta-ketothiolase deficiency (PMID: 31268215). The c.473A>G (p.Asn158Ser) variant affects a highly conserved amino acid; however, in silico tools used to predict the effect of this variant on protein function yield discordant results. This variant has been previously reported as a compound heterozygous or homozygous change in patients with beta-ketothiolase deficiency (PMID: 17236799, 21669895, 27748876, 39519275). A different amino acid change at the same residue (p.Asn158Asp) has also been reported in affected individuals (PMID: 7749408, 17236799). Functional studies indicate this variant leads to reduced catalytic activity and protein instability (PMID: 17236799). The c.473A>G (p.Asn158Ser) variant is present in the latest version of the gnomAD population database at an allele frequency of 0.006% (93/1613970), and is absent in the homozygous state, thus is presumed to be rare. Based on the available evidence, c.473A>G (p.Asn158Ser) is classified as Likely Pathogenic.

ACAT1: PM2, PM3, PM5, PS3:Supporting

The c.473A>G variant in ACAT1 is a missense variant predicted to cause substitution of asparagine to serine at amino acid 158. This variant is rare in the general population with a frequency below the threshold expected for the associated phenotype(s). This variant has been observed in one or more individuals affected with the associated recessive disease, as either homozygous or compound heterozygous with a second variant (PMID: 21669895, 39519275). Additionally, this variant has been observed to segregate in affected family members (PMID: 21669895). This variant has been identified in one or more affected individuals with a phenotype highly consistent with the associated gene (PMID: 21669895, 39519275). A different variant at the same position has been determined to be Pathogenic or Likely Pathogenic. Given the available evidence, this variant is classified as Likely Pathogenic.