Beta-Ketothiolase Deficiency: A Comprehensive Review of Genetic Variants and Pathophysiology
Article information
Abstract
Beta-ketothiolase deficiency (BKD) is a rare autosomal recessive disorder caused by mutations in the ACAT1 gene, also known as mitochondrial acetoacetyl-coenzyme A thiolase (MAT) deficiency. This enzyme defect impairs the breakdown of ketone bodies and isoleucine, leading to significant metabolic disturbances. BKD is characterized by episodic ketoacidosis, developmental delays, muscle weakness, and potential neurological damage, typically manifesting in early childhood. To date, approximately 130 mutations have been reported in association with BKD. This review aims to elucidate the pathophysiological mechanisms underlying the disease and discusses the mutation spectrum of the ACAT1 gene, including its functional implications. Current management strategies emphasize dietary modifications, such as protein restriction and carnitine supplementation, along with vigilant monitoring to prevent metabolic crises and long-term complications. Advances in genetic research, especially the advent of gene therapies like clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, offer promising future treatment options. However, these innovative therapies require extensive research and clinical trials before they can be implemented for BKD patients. Until such advances are realized, genetic counseling remains the cornerstone of preventive care, particularly for families with a history of the disorder or known carrier status.
Introduction
With an estimated prevalence of 1 in 500,000 to 1 in 1,000,000 births, mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (MAT) deficiency is a rare genetic disorder primarily affecting pediatric populations [1]. This disorder is referred to by various names, including alpha-methyl acetoacetic aciduria, 3-ketothiolase deficiency, 3-oxothiolase deficiency, and thiolase (T2) deficiency, reflecting its diverse clinical presentations [2]. First described in 1971, beta-ketothiolase deficiency (BKD) has been documented in approximately 250 cases worldwide to date [3]. Its prevalence varies across ethnic groups, influenced by genetic diversity and carrier frequency within different populations. For example, between 2005 and 2016, 41 BKD cases were identified at a single medical facility in Northern Vietnam, suggesting a probable prevalence of one in 190,000 infants [4]. Newborn screening (NBS) is a critical tool for the early detection of BKD, enabling timely intervention before the onset of severe metabolic complications. In China, from 2009 to 2020, among 16,088,190 screened newborns, only 14 cases were detected through NBS, corresponding to an approximate incidence of one per million infants [5]. These findings highlight the indispensable role of NBS in identifying affected infants, including those who may be asymptomatic at birth.
BKD follows an autosomal recessive inheritance pattern and results from mutations in the ACAT1 gene, which encodes the MAT enzyme responsible for the degradation of isoleucine and the management of ketone bodies. Deficiency of the beta-ketothiolase enzyme impairs the catabolism of specific fatty acids and ketogenic amino acids. When this enzyme is lacking, the body is unable to utilize ketone bodies efficiently for energy, which can lead to serious health complications if left untreated [6]. Severe cases can present with recurrent episodes of metabolic decompensation, manifesting as lethargy, vomiting, hypoglycemia, and metabolic acidosis. If not managed appropriately, these crises may progress to coma or even death. BKD can also provoke metabolic strokes and a range of neurological complications, sometimes resulting in irreversible damage to the basal ganglia, especially in severe cases [7]. Affected individuals may experience developmental delays, muscle weakness, and coordination difficulties. Additionally, the accumulation of certain metabolites can cause a distinctive odor in the breath or sweat [8].
The ACAT1 gene is located on chromosome 11q22.3-23.1, encompassing 12 exons and 11 introns, and spans approximately 14.7 kilobases (kb). It encodes a 427-amino acid precursor protein that includes a 33-amino acid leader peptide, specified by the roughly 1.5 kb human T2 cDNA [4,9]. BKD typically presents between 6 and 18 months of age, often with intermittent episodes of ketoacidosis, although onset at any age is possible [2]. The clinical presentation is variable: some infants may develop symptoms immediately after birth, while others may remain asymptomatic until later in infancy, childhood, or even adulthood. More severe forms tend to manifest earlier and with greater intensity, whereas milder mutations may not present until later in childhood or adulthood. The potential severity of BKD necessitates careful attention to patient outcomes [4,9].
Early detection and intervention are crucial for improving long-term prognosis. Clinical findings typically include marked urinary ketonuria and increased excretion of metabolites such as tiglylglycine, 2-methyl-3-hydroxybutyrate, and 2-methylacetoacetate. However, detecting 2-methylacetoacetate in older urine samples can be challenging. Many countries, including China, Japan, and the United States, have incorporated BKD into NBS programs, utilizing tandem mass spectrometry to detect markers such as tiglylcarnitine (C5:1) and 3-hydroxyisovalerylcarnitine (C5OH). Elevated levels of these markers are frequently observed in BKD patients, although some may exhibit normal acylcarnitine profiles even during metabolic crises. Molecular genetic analysis of the ACAT1 gene is used to confirm the diagnosis [5,10-12].
Numerous investigations have examined the pathophysiological mechanism of BKD in several ethnic groups. Paquay et al. [8] examined 26 French patients born between 1986 and 2014, while Nguyen et al. [4] studied 41 Vietnamese patients born from 2002 to 2016. Abdelkreem et al. [10] reported on 10 MAT patients from Southern India in the same year, and Grunert and Sass [13] studied 32 individuals, most of whom were of European or Turkish ancestry. Abdelkreem et al. [14] provided an updated overview of ACAT1 gene variants and their molecular consequences, reporting data on metabolic decompensation frequency among 221 individuals. Of these, 73 patients (33.0%) experienced multiple acute decompensations, while 198 (89.6%) had at least one metabolic crisis. Specifically, 36 patients (16.3%) had two decompensations, 26 (11.8%) had between three and six episodes, and over nine acute incidents were reported in 1.4% of cases. Notably, 23 individuals (10.4%) were asymptomatic at the time of reporting [10,13,14]. This review has been compiled to provide a comprehensive understanding of the pathophysiology of BKD, the mutation spectrum and functional implications, and an overview of therapeutic strategies for disease management.
Pathophysiological Mechanism of BKD
Mutations in the ACAT1 gene result in deficient activity of MAT, an enzyme essential for the normal functioning of the ketone body metabolism pathway. This deficiency leads to metabolic disturbances, necessitating a closer examination of the thiolytic cleavage pathway for both ketone body utilization and isoleucine catabolism, which play pivotal roles in the pathophysiology of BKD. Thiolytic cleavage is a critical process in ketone body metabolism, particularly in the breakdown of acetoacetate. This enzymatic step, catalyzed by MAT, splits acetoacetyl-CoA into two acetyl-CoA molecules. Acetyl-CoA acts as a central intermediate in cellular energy metabolism and fuels the tricarboxylic acid (TCA) cycle and adenosine triphosphate production. Consequently, impaired MAT activity disrupts the conversion of acetoacetyl-CoA to acetyl-CoA, resulting in the metabolic imbalances characteristic of BKD. This enzymatic reaction enables cells to efficiently utilize ketone bodies as an alternative energy source, which is especially important during periods of fasting, prolonged exercise, or carbohydrate restriction [10].
However, in BKD, deficient MAT activity impairs ketone body metabolism, leading to the clinical and biochemical manifestations of the disorder. Moreover, thiolytic cleavage is not restricted to ketone body metabolism but is also integral to the breakdown of branched-chain amino acids, including isoleucine. Isoleucine is initially converted to α-ketoisocaproate, which then undergoes thiolytic cleavage to yield acetyl-CoA and propionyl-CoA. These metabolites play essential roles in cellular energy metabolism: acetyl-CoA enters the TCA cycle, while propionyl-CoA is further metabolized via the propionate pathway for energy production [13,15-18]. The pathophysiological impact of BKD is summarized in Table 1 and Fig. 1.
Pathophysiological impact of beta-ketothiolase deficiency
Role of mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (MAT) in ketone body metabolism and its therapeutic management. (1) Isoleucine catabolism; (2) MAT deficiency leads to ketoacidosis, metabolite accumulation, and metabolic imbalance (including tiglylglycine [TIG], 2-methyl-3-hydroxybutyrate [2M3HB], and 2-methylacetoacetate [2MAA]); and (3) disorders associated with MAT deficiency in beta-ketothiolase deficiency (BKD). Therapeutic interventions include (4) acute management and (5) long-term management.
The buildup of toxic metabolites in the brain can lead to neuronal dysfunction and damage, resulting in the neurological symptoms observed in affected individuals. The ensuing metabolic acidosis disrupts the body's pH balance, impairing cellular function and causing systemic complications such as electrolyte imbalances, dehydration, and compromised cardiovascular performance. Additionally, impaired energy metabolism leads to muscle weakness and fatigue, particularly during periods of increased physical activity, further contributing to developmental delays and impaired motor function [19]. The liver also plays a crucial role in ketone body metabolism, particularly during fasting or prolonged energy deprivation; thus, impaired ketone body metabolism can result in hepatic dysfunction, manifesting as hepatomegaly and abnormal liver enzyme levels [20].
Mutation Spectrum of the ACAT1 Gene Associated with BKD
Numerous mutations in the ACAT1 gene have been documented across different ethnic groups in association with the development of BKD. By 2010, a total of 70 ACAT1 gene mutations had been reported, most of which are heterogeneous, with many patients carrying unique mutations. Only a small proportion of these mutations have been identified in more than two unrelated families [9]. These mutations include splice site, frameshift, missense, and nonsense variants. Frequently reported mutations include c.278A>G (p.N93S), c.371A>G (p.K124R), c.433C>G (p.Q145E), c.655T>C (p.Y219H), and c.547G>A (p.G183R). These mutations can range in their impact on enzymatic activity from partial to complete loss of function [18].
A pivotal study by Otsuka et al. [21] described a German patient with BKD, which illuminated the complex molecular mechanisms underlying this disorder. The patient, who was only 11 months old at the time of an acute ketoacidosis episode, carried a compound heterozygous mutation: a known null variant c.472A>G (p.N158D) and a previously unreported variant c.949G>A (p.D317N) in the ACAT1 gene. Notably, the c.949G>A variant is located within an exonic splicing enhancer region and was suspected to cause abnormal splicing. Minigene analysis confirmed that this mutation led to partial exon 10 skipping in transcripts. Further investigation revealed that an additional substitution (c.941G>C) could mitigate this effect, underscoring the nuanced interplay of genetic variants. Transient expression studies demonstrated a complete loss of T2 activity in the mutated D317N enzyme, confirming its pathogenicity. This study highlights the potential for both missense mutations and even synonymous substitutions to disrupt enzyme function by interfering with splicing processes, deepening our molecular understanding of BKD [21].
In a significant report by Vakili and Hashemian [1], a 2-month-old girl in Iran presented with fever and toxic encephalopathy, and soon after vaccination developed recurrent episodes of vomiting, hypotonia, seizures, and coma. These episodes persisted until 7 months of age and were characterized by ketoacidosis and the detection of specific urinary metabolites indicating BKD. Genetic analysis of the ACAT1 gene identified a novel homozygous mutation, c.664A>C (p.S222R), marking the first molecularly confirmed case of BKD in Iran [1].
Abdelkreem et al. [14] published a comprehensive overview of 105 ACAT1 gene variants found in 149 patients with BKD. Within this cohort, 56 missense variants were mapped onto the crystal structure of MAT. The majority of these affected residues are either fully or partially buried within the MAT structure, most likely causing BKD through impaired catalytic efficiency and/or reduced folding stability. Furthermore, 30 disease-associated missense mutations were introduced into SV40-transformed human fibroblasts for assessment of their expression and activity. Among these, only two variants (p.C126S and p.Y219H) demonstrated stability comparable to the wild-type enzyme, indicating their potential functional integrity within an otherwise deleterious mutational landscape [14].
In 2020, Manawadu et al. [22] described the first case of BKD in Sri Lanka, characterized by a homozygous c.152C>T (p.P51L) variant. This mutation, classified as a likely pathogenic missense variant according to American College of Medical Genetics and Genomics guidelines, had not been previously reported in exome population databases [22]. Lefevre et al. [23] provided an updated tally of 130 pathogenic variants linked to 250 cases documented in the literature. Among these, the ACAT1 c.622C>T (p.R208*) stop mutation was the most prevalent, identified in at least 36 individuals and observed in both homozygous and compound heterozygous states among Vietnamese, Dutch, and Turkish patients. The major missense variant c.578T>G (p.M193R) was observed in eight Indian families, while the c.1006–1G>C variant, the third most common disease-associated mutation, was identified in 13 families, most of whom were Vietnamese. Additionally, 12 and 10 patients, respectively, were found to have the missense mutation c.949G>A (p.D317N) and two splice site mutations, c.1006-1G>C. The c.949G>A variant, situated within an exonic splice enhancer, leads to exon 10 skipping. Fewer than ten individuals have been reported to carry other ACAT1 gene alterations [13,23].
Lin et al. [5], in a multicenter retrospective study in China, identified five novel variants and proposed 3-hydroxybutyrylcarnitine (C4OH) as a potential marker for BKD screening. Novel variants included c.401T>C (p.M134T) in exon 5, c.481T>C (p.Y161H) in exon 6, c.631C>A (p.Q211K) in exon 7, and c.1119dup (p.V374Sfs86) and c.1154A>T (p.H385L) in exon 11. The c.622C>T (p.R208) variant was the most frequent, with a prevalence of 17.2%, followed by c.1006-1G>C (8.6%) and c.1124A>G (p.N375S) (8.6%). The variants c.997G>C (p.A333P) and c.419T>G (p.L140R) were also notably prevalent [5].
Guo et al. [24] described the development of extra-basal ganglia abnormalities and permanent basal ganglia injury in patients with severe BKD and identified novel mutations, including c.370A>C, c.419T>G (p.L140R), c.373G>T (p.V125F), c.456G>T, and c.72+1G>A. Their study explained the inheritance of compound heterozygous mutations, with each parent contributing a different variant. Further research is needed to confirm the pathogenicity of a potentially novel mutation, c.478C>G (p.P160A, chr11:108009667), due to its predicted impact on protein structure and abundance. Exon 10 skipping was also reported in the variant c.951C>T (p.D317D, chr11:108014720), a synonymous mutation that does not alter the amino acid sequence but may result in a frameshift and nonsense-mediated mRNA decay, thereby contributing to the pathogenesis of BKD [24].
In 2021, Al-Hakami et al. [25] detailed a challenging case of BKD that mimicked type 1 diabetes mellitus. They reported a novel homozygous variant in the ACAT1 gene, designated as c.592G>A (p.Glu198Lys); Chr11(GRCh37):G.108010804G>A [25]. Sun et al. [26] conducted an analysis of ACAT1 gene variants in a child, revealing two compound heterozygous variations: c.121-3C>G and c.275G>A (p.G92D). The c.275G>A variant was identified as de novo, whereas the c.121-3C>G variant was found in the child’s father and sisters. Furthermore, the mother and two sisters were found to carry the c.334+172C>G (rs12226047) polymorphism. Sanger sequencing established that the c.275G>A (p.G92D) and c.334+172C>G (rs12226047) variants were present on the same chromosome. Bioinformatics analysis suggested that both variants were potentially harmful. Ultimately, the c.275G>A mutation was determined to be pathogenic, and the c.121-3C>G variant was classified as likely pathogenic [26].
In 2022, Wang et al. [27] expanded the known mutation spectrum by identifying two previously unreported compound heterozygous variants (c.871G>C and c.1016_1017del) responsible for BKD. These variants were initially detected via whole-exome sequencing and subsequently confirmed by direct sequencing [27]. In 2023, Patra et al. [3] reported a new mutation associated with BKD, presenting with symptoms resembling diabetic ketoacidosis. The mutation was identified as a homozygous ACAT1 variant (c.833T>C, p.V278A) in exon 9 [3].
Zhang et al. [28] analyzed ACAT1 gene mutations in five children and found that the majority of variants (eight out of nine) were missense mutations. Four novel variants were described: c.316C>T (p.Q106*), c.678G>T (p.W226C), c.302A>G (p.Q101R), and c.627_629dupTGA (p.N209_E210insD). The first two mutations were predicted to be pathogenic by SIFT, PolyPhen-2, and Mutation Taster tools. Notably, the nonsense mutation c.316C>T (p.Q106*) resulted in a premature stop codon [28]. Ji et al. [29] published a study on the clinical and genetic diagnosis of three children with isoleucine metabolic disorders, highlighting a novel variant, c.331G>C (p.A111P), thereby expanding the mutational spectrum of BKD. Mutations associated with BKD discovered after 2019 are summarized in Fig. 2, while those identified up to 2019 have been thoroughly reviewed by Abdelkreem et al. [10].
Schematic illustration of the human ACAT1 gene showing the location of 24 variants associated with beta-ketothiolase deficiency reported after 2019. Exons (boxes) and introns (lines) are numbered; c. range above the boxes corresponds to National Center for Biotechnology Information (NCBI) RefSeq: NM_000019.4. Black boxes represent untranslated regions. Exonic variants are indicated above the boxes: black (missense), red (nonsense), and green (other). Mutations located in introns are shown below the diagram.
Therapeutic Interventions
Treatment is systematically divided into two key subcategories: acute management and long-term therapy, both essential for effectively addressing different phases of the disease. In the acute phase, the primary objective is to prevent catabolism and correct dehydration, which are critical elements of emergency intervention. Immediate glucose infusion is pivotal, as it stimulates insulin secretion, suppresses adipocyte lipolysis, and limits the entry of fatty acids into mitochondria for beta-oxidation. In cases of hyperglycemia, glucose administration is reduced, and insulin combined with electrolyte infusion is used to maintain blood glucose levels, support glucose uptake, and reduce fatty acid oxidation, resulting in decreased ketone body production. Severe acidosis is managed with bicarbonate (5%) or bicarbonate bolus (1 mmol/kg) infusions, which increase blood pCO2 and elevate blood pH (>7.1) [30]. This correction of acidosis can cause an intracellular potassium shift, reduce free calcium, and regulate plasma osmolarity. Continuous renal replacement therapy (CRRT) may also be employed to remove accumulated ketone bodies; however, CRRT does not affect ongoing ketone body production, so prolonged use may be necessary [31].
A low-fat, protein-restricted diet (1.5 to 2.0 g/kg) is recommended under the guidance of nutrition therapy. In cases of acute acidosis and carnitine deficiency, L-carnitine infusion (300 to 550 mg/kg/day) and oral L-carnitine supplementation (50 to 200 mg/kg/day) are advised for symptomatic relief [32]. The dosage should be titrated carefully to avoid adverse effects and optimize metabolic support.
Long-term treatment focuses on nutritional management and metabolic monitoring. Patients require a high-energy, protein-restricted diet individualized to their age and clinical condition. The use of specialized formulas and careful monitoring of essential amino acid (isoleucine, leucine, valine) levels are crucial to prevent deficiencies and optimize metabolic control. Dichloroacetate (DCA), a ketone body metabolism inhibitor, may be a potential therapeutic agent for BKD; it is already used in treating enzyme-deficiency diseases such as non-Hodgkin’s lymphoma and lactic acidosis. DCA inhibits the transfer of amino groups from alanine to pyruvate, activates pyruvate oxidation, and thus facilitates alpha-ketoglutarate formation—an early step in isoleucine catabolism in the Krebs cycle. This results in reduced ketone body production and increased insulin-mediated uptake. DCA also helps alleviate refractory metabolic acidosis as a pharmacologic antagonist [33]. Although current therapies provide symptomatic relief, they do not address the underlying genetic defect in ACAT1. Advances in molecular medicine and translational research have made it possible to consider genome editing approaches such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for inborn errors of metabolism [34,35]. This technology may be used to correct pathogenic ACAT1 variants via homology-directed repair or non-homologous end joining [36]. RNA interference using siRNA or miRNA for gene silencing can also increase precision, efficacy, and stability in genetic therapies [37]. Such RNA-based approaches could inhibit the synthesis of ketone body-producing enzymes, including 3-hydroxybutyrate dehydrogenase. The use of ACAT1 mRNA mimics or mRNA delivery via mediated pathways, possibly in combination with other targeted therapies, might offer new strategies for BKD at the molecular level [38,39]. Targeting ACAT1 to block cholesterol esterification has shown potential to enhance CD8+ T-cell function, highlighting its promise as a cancer immunotherapy strategy [40]. However, further research is necessary to translate these experimental therapies into clinical practice.
Discussion and Future Perspectives
BKD is a rare disorder resulting from mutations in the ACAT1 gene. Studies have demonstrated that presymptomatic treatment initiated after detection through NBS leads to better long-term neurodevelopmental outcomes compared to diagnosis following clinical presentation. Therefore, including BKD in routine NBS programs is essential—especially in populations with observed cases or increased carrier frequency—to ensure improved prognosis and prevent life-threatening complications. Currently, there is no cure for BKD. Therapeutic interventions focus on dietary management, supportive care during metabolic crises, and long-term monitoring to prevent neurological complications. Over the past two decades, gene therapy has emerged as a promising treatment strategy for many rare genetic disorders, offering the potential to restore gene function by introducing a normal copy of the defective gene via specific vectors. This approach is being investigated for numerous orphan diseases associated with severe, debilitating phenotypes and limited treatment options. While gene therapy holds the promise of a cure, its availability remains limited to only a few disorders. As for BKD, no gene therapy approaches have yet been attempted. There is a clear need to explore gene therapy for BKD, including the use of viral vector-mediated gene delivery and gene editing platforms such as CRISPR-Cas9. However, until these advanced strategies are developed and tested, genetic counseling remains the gold standard for preventing the birth of affected children. Special attention should be given to families in which both parents are carriers or where there is a history of BKD in a child.
Notes
Conflicts of interest
No potential conflict of interest relevant to this article was reported.
Author contribution
Conceptualization: AK. Data curation: SK. Formal analysis: SK and AK. Funding acquisition: AM. Visualization: AK. Writing - original draft: SK. Writing - review & editing: SK, AK, AC, AK, AW, and AM.
Acknowledgments
Financial support to Sohit Kashyap from DST-PURSE (No IFD/C/I/101123/35/04240), Anil Kumar and Ajay Kumar through ‘Mission Program on Rare Paediatric Genetic Disorders’ PRaGeD (Sanction No-BT/PR45460/MED/12/952/2022) from DBT and Anita Choudhary (Award No- 211610155703) from University Grant Commission (UGC), New Delhi is highly acknowledged. The DST-FIST grant (SR/FST/LS-I/2017/49) to the Department of Human Genetics and Molecular Medicine, Central University of Punjab, is acknowledged with thanks.