Title: Mutation update on <i>ACAT1</i> variants associated with mitochondrial acetoacetyl‐CoA thiolase (T2) deficiency
Abstract: Human MutationVolume 40, Issue 10 p. 1641-1663 MUTATION UPDATEOpen Access Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency Elsayed Abdelkreem, Elsayed Abdelkreem orcid.org/0000-0002-8976-2989 Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan Department of Pediatrics, Faculty of Medicine, Sohag University, Sohag, EgyptSearch for more papers by this authorRajesh K. Harijan, Rajesh K. Harijan Department of Biochemistry, Albert Einstein College of Medicine, New York, New YorkSearch for more papers by this authorSeiji Yamaguchi, Seiji Yamaguchi Department of Pediatrics, Shimane University School of Medicine, Izumo, JapanSearch for more papers by this authorRikkert K. Wierenga, Rikkert K. Wierenga Biocenter Oulu and FBMM, University of Oulu, Oulu, FinlandSearch for more papers by this authorToshiyuki Fukao, Corresponding Author Toshiyuki Fukao [email protected] Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan Correspondence Toshiyuki Fukao, Department of Pediatrics, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu 501-1194, Japan. Email: [email protected] for more papers by this author Elsayed Abdelkreem, Elsayed Abdelkreem orcid.org/0000-0002-8976-2989 Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan Department of Pediatrics, Faculty of Medicine, Sohag University, Sohag, EgyptSearch for more papers by this authorRajesh K. Harijan, Rajesh K. Harijan Department of Biochemistry, Albert Einstein College of Medicine, New York, New YorkSearch for more papers by this authorSeiji Yamaguchi, Seiji Yamaguchi Department of Pediatrics, Shimane University School of Medicine, Izumo, JapanSearch for more papers by this authorRikkert K. Wierenga, Rikkert K. Wierenga Biocenter Oulu and FBMM, University of Oulu, Oulu, FinlandSearch for more papers by this authorToshiyuki Fukao, Corresponding Author Toshiyuki Fukao [email protected] Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan Correspondence Toshiyuki Fukao, Department of Pediatrics, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu 501-1194, Japan. Email: [email protected] for more papers by this author First published: 03 July 2019 https://doi.org/10.1002/humu.23831Citations: 14AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Mitochondrial acetoacetyl-CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine-derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2-deficient patients. The 56 disease-associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease-associated missense ACAT1 variants have been measured by expressing them in human SV40-transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild-type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening. 1 INTRODUCTION The mitochondrial acetoacetyl-CoA thiolase (commonly known as β-ketothiolase [T2]; EC 2.3.1.9; encoded by the ACAT1 gene) is a ubiquitous and important enzyme for ketone body synthesis and degradation as well as in isoleucine catabolism (Fukao et al., 2014, 2018). Human tissues have, at least, five other thiolase isoenzymes: Cytosolic acetoacetyl-CoA thiolase (CT, EC 2.3.1.9), mitochondrial 3-ketoacyl-CoA thiolase (T1, EC 2.3.1.16), the β subunit of the mitochondrial trifunctional enzyme that catalyzes 3-ketoacyl-CoA thiolase activity (TFE, EC 2.3.1.16), the peroxisomal 3-ketoacyl-CoA thiolase (AB-thiolase, EC 2.3.1.16), and the peroxisomal thiolase type-1 (SCP2-thiolase; EC 2.3.1.176). These thiolases (excluding the SCP2-thiolase) share 35–46% sequence identity and have both synthetic and degradative functions; the degradative SCP2-thiolase has very low sequence similarity with any of the other thiolase family members. These thiolases are either dimers (tight dimers) or tetramers (dimers of tight dimers) (Fukao, 2002; Harijan et al., 2013; Kiema et al., 2019). In the biosynthetic direction, thiolases catalyze the formation of a carbon-carbon bond through a Claisen condensation mechanism (from two acetyl-CoA molecules) and in the reverse, degradative direction a C-C bond is broken through thiolysis (in the presence of CoA), resulting in chain shortening of the acyl chain by two carbon atoms (in case the substrate is an unbranched acyl chain) or by three atoms (in case the substrate is a 2-methyl-branched acyl chain), such as for example catalyzed by the T2 (Figure 1; Haapalainen, Meriläinen, & Wierenga, 2006; Song et al., 1994). No cofactors are required for the catalytic activity of thiolases, and each thiolase catalyzes the reaction in both directions. The crystal structures of several thiolases have been reported (Haapalainen et al., 2006; Kiema et al., 2019). From this structural information as well as from extensive sequence alignment, a classification of thiolases has been proposed (Anbazhagan et al., 2014). The crystal structure of the wild-type human T2 thiolase tetramer has been reported in 2007 (Haapalainen et al., 2007). Two cysteines are important for catalysis. The nucleophilic cysteine (Cys126 in human T2 thiolase) becomes acetylated in the reaction cycle (Figure 1), whereas the second catalytic cysteine (Cys413) functions as an acid/base (Figure 2). These cysteines protrude into the catalytic site from two different catalytic loops, being the CxS loop and the CxG loop (Figure 2 and Figure 3). Figure 1Open in figure viewerPowerPoint The reactions catalyzed by the T2 thiolase. (a) The biosynthetic reaction: The substrates are two molecules of acetyl-CoA. (b) The degradative reaction: The substrates are 2-methylacetoacetyl-CoA (or acetoacetyl-CoA) and CoA. In both directions, the reaction mechanism proceeds via a covalent intermediate, in which the nucleophilic cysteine, Cys126 in human T2, becomes acetylated in the biosynthetic as well as in the degradative reactions Figure 2Open in figure viewerPowerPoint Schematic drawing showing the T2 thiolase reaction in the synthetic direction. Two molecules of acetyl-CoA are converted into CoA and acetoacetyl-CoA. The role of the four catalytic residues (Cys126, Asn353, His385, Cys413 of human T2) is highlighted. These residues protrude into the catalytic site from the four catalytic loops (the CxS, NEAF, GHP, and CxG loops, respectively, shown in bold). Cys126 is the nucleophilic cysteine and Cys413 is the acid/base cysteine. The side chains of Asn353 (fixing Wat98) and His385, as well as the main chain N-atoms of the CxS and CxG loops, contribute to the two oxyanion holes (OAH1 and OAH2, shown as shaded semicircles). These oxyanion holes stabilize the negative charge that develops during the reaction on the thioester oxygen atom of the reaction intermediates, being therefore also critically important for catalysis. The short-curved arrows visualize the breaking/forming of bonds Figure 3Open in figure viewerPowerPoint The sequence of the human mitochondrial acetoacetyl-CoA thiolase (T2, UniProt code: P24752) with nomenclature of secondary structure, sequence fingerprints, and loops. The N-terminal region is the mitochondrial leader sequence, which is cleaved off on entry into the mitochondria. The secondary structure is obtained from the structure of the human T2 (PDB code: 2IBW) using the ESPript 3.0 server (Robert & Gouet, 2014) and shown above the sequence. An asterisk (*) above the sequence marks every tenth residue. The mature sequence starts at Val34, indicated by a black circle (•) above the sequence. Important active site loops that are near the catalytic site are identified below the sequence with their sequence fingerprint. The nomenclature of the functional regions of the loop domain (residues 156–286) is also given below the sequence. The structural properties of the latter loop regions are visualized in Figure 7 and Figure S3 Ketone bodies (acetoacetate and 3-hydroxybutyrate) are important energy sources for most tissues, particularly the brain. Ketone body synthesis begins in the liver by β-oxidation of free fatty acids to output acetyl-CoA and acetoacetyl-CoA. T2 in the liver catalyzes the Claisen condensation of two acetyl-CoA molecules into acetoacetyl-CoA. In extrahepatic tissues, T2 is responsible for thiolytic cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA. T2 deficiency causes episodic ketoacidosis. This indicates that T2 deficiency impedes ketolysis to a greater extent than ketogenesis. The abundant amount of T1 in the liver likely compensates for T2 deficiency in ketogenesis (Fukao et al., 2014). Potassium ions specifically enhance the activity of T2 but do not change that of T1 and other thiolases, therefore the potassium ion-activated acetoacetyl-CoA thiolase assay remains the gold-standard test for the T2 enzyme assay (Middleton, 1973). In isoleucine catabolism, T2 catalyzes the thiolysis of 2-methylacetoacetyl-CoA (2MAA-CoA) to acetyl-CoA and propionyl-CoA. T2 deficiency is characterized by excessive accumulation of isoleucine-catabolic intermediates that can be detected in urine as 2-methylacetoacetate (2MAA), 2-methyl-3-hydroxybutyrate (2M3HB), and tiglyl-glycine (TIG) and in blood as tiglyl-carnitine and 2M3HB-carnitine; notably, 2MAA is rapidly degraded and, consequently, is sometimes hardly detected in urine samples, especially in nonfresh ones (Aramaki et al., 1991). Therefore, T2 deficiency results in excessive accumulation of not only 2MAA-CoA but also of the two upstream metabolites, namely 2M3HB-CoA and 2-methyl-2E-butenoyl-CoA (tiglyl-CoA) (Fukao et al., 2014). T2 deficiency (MIM# 203750, 607809) is an autosomal recessive disease. Deficiencies of T2 and 3-hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4; MIM# 246450) constitute the most common inborn errors of ketone body metabolism (Abdelkreem et al., 2016; Fukao et al., 2014). Since Daum, Lamm, Mamer, and Scriver (1971), for the first time, characterized T2 deficiency, at least 159 patients (Supporting Information Table) with the disease have been confirmed (through enzyme assay and/or genetic analysis) worldwide without ethnic preference. The incidence of T2 deficiency has been estimated in some regions, as one per 232,000 newborns in Minnesota, one per 190,000 newborns in northern Vietnam, and one per 111,000 newborns in Hyderabad (India) (Abdelkreem, Akella, et al., 2017; Nguyen et al., 2017; Sarafoglou et al., 2011). Herein, we review 105 ACAT1 variants that have been reported in 149 patients with T2 deficiency; we use the term "disease-associated ACAT1 variants" to refer to variants associated with T2 deficiency. A discussion on non-disease-associated ACAT1 variants is beyond the scope of this review. We discuss important structural features of human T2 and the location of the disease-associated missense ACAT1 variants in the context of the crystal structure of human T2. To increase the understanding of this rare disease, we also discuss its clinical and laboratory implications. 2 THE T2 GENE AND DISEASE-ASSOCIATED VARIANTS The human ACAT1 gene (NCBI reference sequence: NG_009888.1) is located on chromosome 11q22.3-q23.1, spanning approximately 27 kb. This gene contains 12 exons interspersed by 11 introns. The 5ʹ-flanking region lacks a classic TATA box, but it contains two CAAT boxes and is GC rich. These features are characteristic of housekeeping genes. Human T2 complementary DNA (cDNA; NCBI reference sequence: NM_000019.3) spans about 1.5 kb. It encodes a precursor protein (NCBI reference sequence: NP_000010.1) composed of 427 amino acids, including a leader polypeptide of 33-amino acid (Kano et al., 1991). The sequence of human T2 is shown in Figure 3. The available data on ACAT1 variants associated with T2 deficiency are shown in three tables. Table 1, 2 have the information on the disease-associated missense variants. These two tables also describe information on the location of the variant site with respect to the structure, in particular, whether the side chain of a residue is buried or whether it is exposed to bulk solvent. Table 1 lists the missense variants that have also been characterized with respect to (a) expression efficiency and (b) catalytic activity properties. The experimental details related to these characterizations are provided in the Supporting Information. For some variants, this information is available for expression at three temperatures; 30, 37, and 40°C. As can be seen in Table 1, there is generally a good correlation between the results obtained at different temperatures (e.g., whenever the data of expression at three temperatures are available, then the expression levels are the highest at 30°C and the lowest at 40°C). In addition, the activity recovery is generally never higher than the expression recovery. Table 3 lists other disease-associated variants (ATG initiation codon, insertions, deletions, duplications, nonsense and aberrant splicing). Figure 4 depicts the location of the disease-associated variants with respect to the exons of the ACAT1 gene. Table 1. Missense ACAT1 variants associated with T2 deficiency, with available expression and activity data (n = 30) E/I Nucleotide changea Predicted amino acid changea In silico prediction of pathogenicity Enzyme assayd/Expression assaye by expression at References Comments on the structural information Involvement of glycine or proline in the mutation Important properties of each residue with respect to the structure of the tetramerf: PolyPhen-2 scoreb SIFT scorec 37°C (in bold if equal or higher than 25% expressed) 30°C 40°C -Buried (completely buried) -Surface (partially buried) -Exposed side chain (side chain points towards solvent) E3 c.218A>C p.Gln73Pro 0.72 0.03 0%/0% 0%/0% 0%/0% Sakurai et al. (2007) Q73P Surface E4 c.278A>G p.Asn93Ser 0.85 0.04 8%/60% NM NM Fukao et al. (1998), Fukao, Zhang, et al. (2003) At the dimer interface, maybe expressed as a folded monomer? Buried E5 c.371A>G p.Lys124Arg 1.00 0.03 0%/0% 0%/0% NM Fukao et al. (2001), Fukao, Nakamura, et al. (2002) At the dimer interface Buried E5 c.377G>C p.Cys126Ser 0.99 0.00 0%/100% NM NM This paper Side chain points towards the catalytic site Exposed side chain E5 c.380C>T p.Ala127Val (6% of mRNA), activates cryptic splice acceptor site causing c.336_386 del (p.Leu113_Gly129 del) (94% of mRNA) 0.98 0.01 0%/12% 0%/50% NM Nakamura et al. (2001), Fukao, Nakamura, et al. (2002) Buried E5 c.395C>G p.Ala132Gly 0.11 0.02 10%/10% 25%/25% NM Zhang et al. (2004) A132G Buried E5 c.431A>C p.His144Pro 0.47 0.23 25%/50% NM 25%/NM Fukao et al. (2012) At the dimer interface, maybe expressed as a folded monomer? H144P Surface E5 c.433C>G p.Gln145Glu 0.98 0.76 15%/12% 30%/25% NM Riudor et al. (1995), Fukao et al. (2001), Fukao, Nakamura, et al. (2002) At the dimer interface, maybe expressed as a folded monomer? Surface E6 c.455G>C p.Gly152Ala 1.00 0.00 0%/0% 5%/25% NM Zhang et al. (2004), Fukao et al. (2001), Fukao, Nakamura, et al. (2002), Buhaş et al (2013), Paquay et al. (2017) G152A Buried E6 c.472A>G p.Asn158Asp 0.93 0.05 0%/5% 0%/50% 0%/0% Wakazono et al. (1995), Fukao, Yamaguchi, et al. (1995); Fukao et al. (2001), Sakurai et al. (2007), Buhaş et al. (2013), Otsuka et al. (2016) At the dimer interface Surface E6 c.473A>G p.Asn158Ser 0.80 0.12 0%/2% 0% / 3% 0%/0% Sakurai et al. (2007), Sarafoglou et al. (2011) At the dimer interface Surface E6 c.556G>T p.Asp186Tyr 1.00 0.00 0%/33% 0%/NM NM Fukao, Horikawa, et al. (2010), Hori et al. (2015) In the covering loop Buried E6 c.578T>G p.Met193Arg 0.99 0.00 0%/0% 0%/0% 0%/0% Ali et al. (2011), Akella et al. (2014), Abdelkreem, Akella, et al. (2017), Grünert et al. (2017) Side chain points towards the pantetheine binding tunnel. Exposed side chain E7 c.623G>A p.Arg208Gln 1.00 0.00 0%/50% 0%/60% 0%/50% Sakurai et al. (2007) Side chain fixes the adenine binding loop Exposed side chain E7 c.643_644delinsAA p.Ala215Asn 1.00 0.00 0%/0% 0%/0% 0%/0% Abdelkreem, Akella, et al. (2017) Buried E7 c.655T>C p.Tyr219His 1.00 0.13 0%/100% 0%/100% 0%/50% Fukao et al. (2001), Sakurai et al. (2007) Side chain interacts with the potassium ion and the CoA moiety Exposed side chain E7 c.674C>A p.Ala225Glu 1.00 0.00 0%/0% NM NM Abdelkreem, Alobaidy, et al. (2017) Surface E8 c.759T>A p.Asp253Glu 1.00 0.35 0%/0% NM NM Fukao et al. (2001), this paper Near the cationic loop Buried E9 c.844A>C p.Asn282His 1.00 0.00 0%/50% 0%/100% 0%/40% Sakurai et al. (2007) In the pantetheine loop Buried E9 c.890C>T p.Thr297Met 1.00 0.00 10%/12% 20%/20% NM Wakazono et al. (1995), Fukao et al. (2001), Zhang et al. (2004) Surface E9 c.901G>C p.Ala301Pro 0.99 0.02 0%/10% NM NM Wakazono et al. (1995) A301P Buried E9 c.935T>C p.Ile312Thr 0.96 0.00 8%/8% NM NM Fukao et al. (1998), (2001), Fukao, Zhang, et al. (2003) Buried E10 c.949G>A p.Asp317Asn (≈20% of mRNA), affects ESE sequence causing exon 10 skipping (≈80% of mRNA) 1.00 0.05 0%/0% NM NM Otsuka et al. (2016), Köse et al. (2016), Grünert et al. (2017), this paper Surface E10 c.968T>C p.Ile323Thr 0.87 0.67 20%/25% 40%/40% 0%/0% Abdelkreem, Akella, et al. (2017) In the Cβ1-Cα1 loop that shapes the binding pocket of the 2-methyl group of the 2-methylacetoacetyl-CoA substrate and side chain interacts with the covering loop Exposed side chain E10 c.997G>C p.Ala333Pro 0.99 0.01 0%/0% NM NM Fukao et al. (1998), (2001), Fukao, Zhang, et al. (2003) A333P Surface E11 c.1059T>A p.Asn353Lys 1.00 0.00 0%/0% 0%/0% 0%/0% Sakurai et al. (2007) In the NEAF loop Buried E11 c.1061A>T p.Glu354Val 1.00 0.00 0%/0% 0%/0% NM Fukao et al. (2001), Fukao, Nakamura, et al. (2002) In the NEAF loop Buried E11 c.1124A>G p.Asn375Ser (11% of mRNA), activates a cryptic splice donor site causing c.1120_1163del (89% of mRNA) 1.00 0.00 0%/0% 0%/0% NM Fukao et al. (2008), Abdelkreem, Akella, et al. (2017) Buried E12 c.1168T>C p.Ser390Pro 1.00 0.00 0%/0% NM 0%/NM Fukao et al. (2012) S390P Buried E12 c.1189C>G p.His397Asp 0.99 0.12 0%/0% 0%/0% NM Zhang et al. (2004), Catanzano et al. (2010), Paquay et al. (2017) Buried Abbreviations: E, exon; ESE, exonic splicing enhancer; I, intron; mRNA, messenger RNA; NM, not measured; T2, mitochondrial acetoacetyl-CoA thiolase a Description of nucleotide changes, exons/introns, and predicted amino acid change follows the HGVS nomenclature (version 15.11, http://varnomen.hgvs.org; den Dunnen et al., 2016) using ACAT1 NCBI reference sequences (NM_000019.3, NG_009888.1, and NP_000010.1) with +1 as the number of the A of the ATG initiation codon. b PolyPhen-2 (polymorphism phenotyping v2; http://genetics.bwh.harvard.edu/pph2/) is a tool that predicts the effect of an amino acid substitution on protein structure and function. Score ranges from 0 to 1; higher scores predict an increased possibility for a damaging effect. A predicted benign value is shown in bold. c SIFT (sorting intolerant from tolerant; https://sift.bii.a-star.edu.sg/) is a sequence homology-based tool that predicts the effect (damaging if the score is ≤0.05 and tolerated if the score is >0.05) of an amino acid substitution on protein function. Seven predicted tolerant values (score >0.05) are shown in bold. d Percentage of catalytic activity with respect to wild-type T2 control, using potassium-activated acetoacetyl-CoA thiolase assay (Supporting Information Material). e Percentage of expressed soluble protein with respect to wild-type T2 control (Material S2). f The classification is from visual inspection of the tetramer. The PDB code of the reference structure is 2IBW. This structure is the complex of human T2-thiolase complexed with CoA, K+ and Cl−. For the classification, the unliganded structure (without CoA, K+, Cl−) has been considered. Table 2. Missense ACAT1 variants associated with T2 deficiency, with no available expression and activity data (n = 26) E/I Nucleotide changea Predicted amino acid changea References Comments on the structural information Involvement of glycine or proline in the mutation Important properties of each residue with respect to the structure of the tetramerb: -Buried (completely buried) -Surface (partially buried) -Exposed side chain (side chain points towards solvent) E4 c.299G>A p.Gly100Glu Wojcik et al. (2018) At the dimer interface G100Q Surface E4 c.301C>A p.Gln101Lys Grünert et al. (2017) At the dimer interface Surface E5 c.370A>G p.Lys124Glu Grünert et al. (2017) At the dimer interface Buried E6 c.460G>A p.Glu154Lys Ali et al. (2011) Buried E6 c.534G>T p.Leu178Phe Paquay et al. (2017) At the dimer interface Buried E6 c.547G>A p.Gly183Arg Fukao, Yamaguchi, Orii, Schutgens, et al. (1992), Fukao et al. (2001), Grünert et al. (2017), Hu et al. (2017) At the dimer interface G183R Buried E6 c.578T>C p.Met193Thr Mrázová et al. (2005), Thümmler et al. (2010) Side chain points towards the pantetheine binding tunnel Exposed side chain E7 c.602C>T p.Ala201Val Fukao et al. (2013) Buried E7 c.653C>T p.Ser218Phe Wen et al. (2016) Buried E7 c.664A>C p.Ser222Arg Vakili and Hashemian (2018) Buried E8 c.760G>A p.Glu254Lys Paquay et al. (2017) Just after the cationic loop, side chain fixes the Nβ1-Nα1 loop Exposed side chin E8 c.764A>C p.Glu255Ala Sundaram, Nair, Namboodhiri, and Menon (2018) This residue is just after the cationic loop Surface E8 c.765A>T p.Glu255Asp Paquay et al. (2017) This residue is just after the cationic loop Surface E9 c.829A>C p.Thr277Pro Su et al. (2017) T277P Surface E9 c.851G>A p.Ser284Asn Nguyen et al. (2017) In the pantetheine binding loop Surface E9 c.854C>T p.Thr285Ile Al-Shamsi, Hertecant, Al-Hamad, Souid, and Al-Jasmi (2014) In the pantetheine binding loop Surface E9 c.890C>A p.Thr297Lys Su et al. (2017) Surface E11 c.1040T>C p.Ile347Thr Mrázová et al. (2005), Grünert et al. (2017) Buried E11 c.1059T>G p.Asn353Lys Paquay et al. (2017) Part of the NEAF motif Buried E11 c.1136G>T p.Gly379Val Fukao et al. (1994), (2001) G379V Buried E11 c.1138G>A p.Ala380Thr Fukao et al. (1991), (2001) Buried E11 c.1160T>C p.Ile387Thr Wojcik et al. (2018) Side chain points towards the catalytic site. Exposed side chin E11 c.1163G>A p.Gly388Glu, splice donor site with probable exon 11 skipping Paquay et al. (2017) G388E Buried E12 c.1167G>A p.Met389Ile Paquay et al. (2017) Buried E12 c.1229C>T p.Ala410Val Nguyen et al. (2015), (2017) Buried E12 c.1253G>A p.Gly418Asp Grünert et al. (2017) At the dimer interface, interacts with the chloride ion G418D Buried Abbreviations: E, exon; I, intron; T2, mitochondrial acetoacetyl-CoA thiolase a Description of nucleotide changes, exons/introns, and predicted amino acid change follows the HGVS nomenclature (version 15.11, http://varnomen.hgvs.org; den Dunnen et al., 2016) using ACAT1 NCBI reference sequences (NM_000019.3, NG_009888.1, and NP_000010.1) with +1 as the number of the A of the ATG initiation codon. b The classification is from visual inspection of the tetramer. The PDB code of the reference structure is 2IBW. This structure is the complex of human T2-thiolase complexed with CoA, K+ and Cl−. For the classification, the unliganded structure (without CoA, K+, Cl−) has been considered. Table 3. Other ACAT1 variants associated with T2 deficiency (n = 49) E/I Nucleotide changea Predicted amino acid changea Reference (A) ATG initiation codon (n = 3) E1 c.1A>G Reduced translation efficiency (11%) Fukao, Matsuo, et al. (2003), Nguyen et al. (2017) E1 c.2T>A Reduced translation efficiency (7.4%) Fukao et al. (1993), Fukao, Matsuo, et al. (2003) E1 c.2T>C Reduced translation efficiency (19%) Fukao, Zhang, et al. (2003), Fukao, Matsuo, et al. (2003) (B) In-frame deletions/insertions/duplications (n = 7) E3 c.163_167delinsAA p.Phe55_Leu56delinsLys Fukao, Nguyen, et al. (2010), Nguyen et al. (2015), (2017) E4 c.254_256del p.Glu85del Fukao, Nakamura, et al. (2002) E8 c.756_758del p.Glu252del Sakurai et al. (2007) E10 c.947_949dup p.Ala316dup Paquay et al. (2017) E11 c.1016_1018dup p.Asp339dup Zhang et al. (2004), Paquay et al. (2017) E11 c.1035_1037del p.Glu345del Sewell et al. (1998), Fukao et al. (2001) E12 c.1241_1245delinsGT p.Asn414_Gly415delinsSer Gibson, Elpeleg, and Bennett (1996), this paper (C) Out-of-frame deletions/insertions/duplications, nonsense, aberrant splicing, others (n = 39) E1 c.52dup p.Leu18Profs*49 Zhang et al. (2004), Sarafoglou et al. (2011), Paquay et al. (2017) E2 c.79A>T p.Arg27* Paquay et al. (2017) E2 c.83_84del p.Tyr28Cysfs*38 Fukao et al. (1997), Paquay et al. (2017), Su et al. (2017) E2 c.86_87dup p.Glu30Trpfs*11 Al-Shamsi et al. (2014), Al-Jasmi, Al-Shamsi, Hertecant, Al-Hamad, and Souid (2016) E2 c.99T>A p.Tyr33* Fukao, Yamaguchi, et al. (1995), Fukao et al. (2001) I2 c.121–3C>G Splice acceptor site (probably exon 3 skipping) Su et al. (2017) I2 c.121–13T>A Splice acceptor site (causing exon 3 skipping in >90% of mRNA) Aoyama et al. (2017) E3 c.149del p.Thr50Asnfs*7 Fukao et al. (1998), (2001), Fukao, Zhang, et al. (2003), Hori et al. (2015) E4 c.286C>T p.Gln96* Sarafoglou et al. (2011) I4 c.334+1G>A splice donor site (probably exon 4 skipping) Grünert et al. (2017) E5 c.354_355delinsG p.Cys119Valfs*4 Law et al. (2015) E5 c.414_415del p.Leu140Tyrfs*36 Paquay et al. (2017) I5 c.435+1G>A splice donor site (probably exon 5 skipping) Fukao et al. (1997) E6 c.446del p.Val149Glyfs*14 Paquay et al. (2017) E6 c.462_482delinsTCCTC p.Glu154Aspfs*4 Grünert et al. (2017) E7 c.622C>T p.Arg208* Fukao, Nguyen, et al. (2010), Sarafoglou et al. (2011), Wen et al. (2016), Nguyen et al. (2015), (2017), Grünert et al. (2017) I7 c.730+1G>A Splice donor site (probably exon 7 skipping) Abdelkreem, Akella, et al. (2017) I7-E8 c.731–46_752del Splice acceptor site (causing exon 8 skipping) Fukao, Song, et al. (1995), Fukao, Yamaguchi, et al. (1995), (2001) E8 c.754_755insCT p.Glu252Alafs*17 Fukao et al. (1997), (2001) E8 c.814C>T p.Gln272* (75% of mRNA), affects ESE sequence causing exon 8 skipping (25% of mRNA) Fukao et al. (1994), Sakurai et al. (2007), Paquay et al. (2017) I8 c.826+1G>T Splice donor site (causing exon 8 skipping) Fukao, Yamaguchi, Orii, Schutgens, et al. (1992), (2001), Wakazono et al. (1995), Zhang et al. (2004), Paquay et al. (2017), Grünert et al. (2017) I8 c.826+5G>T Splice donor site (causing exon 8 skipping) Thümmler et al. (2010) I8 c.826+5_826+9del Splice donor site (probably exon 8 skipping) Grünert et al. (2017) I9 c.940+1G>T Splice donor site (probably exon 9 skipping) Grünert et al. (2017) I9 c.941–9T>A Splice acceptor site (causing exon 10 skipping in 90% of transcripts) Sasai et al. (2017) E10 c.951C>T Affects ESE sequence causing exon 10 skipping (≈ 40% of mRNA) Fukao, Horikawa, et al. (2010), Otsuka et al. (2016) p.317Asp = (≈ 60% of mRNA) I10 c.1006–2A>C Splice acceptor site (causing exon 11 skipping) Fukao, Yamaguchi, Orii, Schutgens, et al. (1992), (2001), Wojcik et al. (2018) I10 c.1006–1G>C Splice acceptor site (causing exon 11 skipping) Fukao, Yamaguchi, Orii, Osumi, et al. (1992), (2001), Nguyen et al. (2015), (2017), Su et al. (2017), Wojcik et al. (2018) I10 c.1006–1G>A Splice acceptor site (probably exon 11 skipping) Law et al. (2015) E11 c.1013_1016dup p.Asp339Glufs*17 Abdelkreem, Akella, et al. (2017) E11 c.1032dup p.Glu345Argfs*10 Nguyen et al. (2015), (2017) E11 c.1033_1034del p.Glu345Argfs*9 Paquay et al. (2017) E11 c.1083dup p.Ala362Serfs*4 Sewell et al. (1998), Fukao et al. (2001) I11 c.1163+2T>C Splice donor site (activates cryptic splice site causing c.1163_1164ins GCAG) Fukao et al. (1993), (2001), Grünert et al. (2017) E12 c.1223_1226dup p.Ala410Serfs*51 Paquay et al. (2017) g.20623_29833delinsGTAA Probably del exons 6–11 Nguyen et al. (2017) c.(120+1_121-1)_(344+1_345-1)del del exons 3–4 Fukao et al. (2013) c.(72+1_73-1)_(344+1_345-1)del, c.(72+1_73-1)_(435+1_436-1)del del exons 2–4 (≈ 10% of mRNA), del exons 2–5 (≈ 90% of mRNA) Zhang et al. (2006) c.(730+1_731-1)_(940+1_941-1)dup Tandem duplication of exons 8–9 Fukao et al. (2007) Abbreviations: E, exon; ESE, exonic splicing enhancer; I, intron; T2, mitochondrial acetoacetyl-CoA thiolase a Description of nucleotide changes, exons/introns, and predicted amino acid change follows the HGVS nomenclature (version 15.11, http://varnomen.hgvs.or