[Clinical analysis and genetic diagnosis of three children with Isoleucine metabolic disorders due to variants of HSD17B10 and ACAT1 genes].

OBJECTIVE: To explore the clinical, biochemical and genetic characteristics of three children with Isoleucine metabolic disorders due to variants of HSD17B10 and ACAT1 genes. METHODS: Two children with 17ß hydroxysteroid dehydrogenase 10 (HSD17B10) deficiency and a child with ß-ketothiolase deficiency (BKD) diagnosed at Shanghai Children's Hospital between 2014 and 2021 were selected as the study subjects. Clinical data of the children were collected. The children were subjected to blood acylcarnitine, urinary organic acid and genetic testing, and candidate variants were analyzed with bioinformatic tools. RESULTS: The main symptoms of the three children had included epilepsy, developmental delay, hypotonia and acidosis. Their blood acylcarnitine methylcrotonyl carnitine (C5:1), 3-hydroxyisovalerylcarnitine (C5-OH) and 3-hydroxybutylcarnitine (C4OH) were increased to various extents, and urine organic acids including methyl crotonylglycine and 2-methyl-3-hydroxybutyric acid were significantly increased. Child 1 and child 2 were respectively found to harbor a c.347G>A (p.R116Q) variant and a c.274G>A (p.A92T) variant of the HSD17B10 gene, and child 3 was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.547G>A (p.G183R) and a c.331G>C (p.A111P). Among these, the c.274G>A (p.A92T) and c.331G>C (p.A111P) variants were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), they were respectively classified as variant of unknown significance (PP3_Strong+PM2_supporting) and likely pathogenic (PM3+PM2_Supporting+PP3_Moderate+PP4). CONCLUSION: Both the HSD17B10 deficiency and BKD can lead to Isoleucine metabolism disorders, which may be difficult to distinguish clinically. Genetic testing can further confirm the diagnosis. Discoveries of the HSD17B10: c.274G>A (p.A92T) variant and the ACAT1: c.331G>C (p.A111P) variant have enriched the mutational spectrum of the two diseases.

[Clinical features and genetic analysis of three children with ß-ketothiolase deficiency].

OBJECTIVE: To explore the clinical features and genetic variants in three children suspected for ß-ketothiolase deficiency (BKTD). METHODS: Clinical manifestations, laboratory examination and genetic testing of three children suspected for BKTD at Henan Children's Hospital between January 2018 and October 2022 were collected, and their clinical and genetic variants were retrospectively analyzed. RESULTS: The children were all males with a age from 7 to 11 months. Their clinical manifestations have included poor spirit, shortness of breath, vomiting, convulsions after traumatic stress and/or infection. All of them had severe metabolic acidosis, elevated ketone bodies in blood and urine, hypoglycemia, with increased isoprenyl-carnitine and 3-hydroxyisovalyl-carnitine in the blood, and 2-methyl-3-hydroxybutyrate and methylprotaroyl glycine in the urine. All of them were found to harbor compound heterozygous variants of the ACAT1 gene, including c.1183G>T and a large fragment deletion (11q22.3-11q23.1) in child 1, c.121-3C>G and c.826+5_826+9delGTGTT in child 2, and c.928G>C and c.1142T>C in child 3. The variants harbored by children 2 and 3 were known to be pathogenic or likely pathogenic. The heterozygous c.1183G>T variant in child 1 was unreported previously and rated as a variant of unknown significance (PM2_Supporting+PP3+PP4) based on guidelines from the American College of Medical Genetics and Genomics. The large segment deletion in 11q22.3-11q23.1 has not been included in the DGV Database and was rated as a pathogenic copy number variation. CONCLUSION: The variants of the ACAT1 gene probably underlay the pathogenesis of BKTD in these three children.

Identification of two novel ACAT1 variant associated with beta-ketothiolase deficiency in a 9-month-old boy.

OBJECTIVES: Mitochondrial acetoacetyl-CoA thiolase (beta-ketothiolase, T2) is necessary for the catabolism of ketone bodies andisoleucine. T2 deficiency is an autosomal recessive metabolic disorder caused by variant in the ACAT1 gene. In this report, we describe two novel ACAT1 variant identified in a Chinese family. CASE PRESENTATION: The 9-month-old male proband was admitted to the pediatric intensive care unit for altered consciousness. At the time of admission, the patient had acidosis, drowsiness, and respiratory failure. Both urine organic acid analyses and LC-MS/MS suggested T2 deficiency. Novel compound heterozygous variant (c.871G>C and c.1016_1017del) in the ACAT1 gene were detected in the proband by WES and verified through direct sequencing. Family analysis demonstrated that the first variant was transmitted from his father and the second variant was from his mother, indicating autosomal recessive inheritance. This report is the first to describe the association of these variant with T2 deficiency based on genetic testing. Although these variant were identified in the patient's elder sister and elder brother, they continue to be asymptomatic. CONCLUSIONS: We identified two novel ACAT1 variants associated with T2 deficiency. The identification expands the spectrum of known variant linked to the disorder.

C4OH is a potential newborn screening marker-a multicenter retrospective study of patients with beta-ketothiolase deficiency in China.

BACKGROUND: Beta-ketothiolase deficiency (BKTD) is an autosomal recessive disorder caused by biallelic mutation of ACAT1 that affects both isoleucine catabolism and ketolysis. There is little information available regarding the incidence, newborn screening (NBS), and mutational spectrum of BKTD in China. RESULTS: We collected NBS, biochemical, clinical, and ACAT1 mutation data from 18 provinces or municipalities in China between January 2009 and May 2020, and systematically assessed all available published data from Chinese BKTD patients. A total of 16,088,190 newborns were screened and 14 patients were identified through NBS, with an estimated incidence of 1 per 1 million newborns in China. In total, twenty-nine patients were genetically diagnosed with BKTD, 12 of which were newly identified. Most patients exhibited typical blood acylcarnitine and urinary organic acid profiles. Interestingly, almost all patients (15/16, 94%) showed elevated 3-hydroxybutyrylcarnitine (C4OH) levels. Eighteen patients presented with acute metabolic decompensations and displayed variable clinical symptoms. The acute episodes of nine patients were triggered by infections, diarrhea, or an inflammatory response to vaccination. Approximately two-thirds of patients had favorable outcomes, one showed a developmental delay and three died. Twenty-seven distinct variants were identified in ACAT1, among which five were found to be novel. CONCLUSION: This study presented the largest series of BKTD cohorts in China. Our results indicated that C4OH is a useful marker for the detection of BKTD. The performance of BKTD NBS could be improved by the addition of C4OH to the current panel of 3-hydroxyisovalerylcarnitine and tiglylcarnitine markers in NBS. The mutational spectrum and molecular profiles of ACAT1 in the Chinese population were expanded with five newly identified variants.

[Analysis of ACAT1 gene variants in a patient with ß-ketothiolase deficiency].

OBJECTIVE: To explore the genetic etiology of a child suspected for ß-ketothiolase deficiency by neonatal screening. METHODS: All coding exons and flanking sequences of the ACAT1 gene were subjected to targeted capture and high-throughput sequencing. Suspected variants were verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.121-3C>G and c.275G>A (p. Gly92Asp). The c.121-3C>G variant was also detected in his father and two sisters, while the c.275G>A (p. Gly92Asp) was a de novo variant. A c.334+ 172C>G (rs12226047) polymorphism was also detected in his mother and two sisters. Sanger sequencing has verified that the c.275G>A (p. Gly92Asp) and c.334+172C>G (rs12226047) variants are located on the same chromosome. Bioinformatics analysis suggested both c.121-3C>G and c.275G>A (p.G92D) variants to be damaging. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.275G>A variant of the ACAT1 gene was predicted to be pathogenic (PS2+ PM2+ PM3+ PP3+PP4), the c.121-3C>G variant to be likely pathogenic (PM2+ PM3+ PP3+PP4). CONCLUSION: The c.121-3C>G and c.275G>A variants of the ACAT1 gene probably underlay the pathogenesis of the child. Above finding has enriched the variant spectrum of the ACAT1 gene.

Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency.

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.

Beta-ketothiolase deficiency in a Malaysian infant.

Methylacetoacetyl-coenzyme A thiolase (MAT) deficiency is an autosomal recessive disease caused by a defect of mitochondrial acetoacetyl-CoA thiolase (T2). There is an error of isoleucine catabolism and ketone body utilization due to mutations in the acetyl-Coenzyme A acetyltransferase 1 (ACAT1) gene. We report a case of a 14 months old Sabahan boy with beta deficiency who presented with severe sepsis and ketoacidosis who subsequently recovered.

[Retrospective analysis on clinical data and genetic variations of patients with beta-ketothiolase deficiency].

OBJECTIVE: To summarize the clinical, biochemical and molecular characteristics of 8 patients with beta-ketothiolase deficiency (BKD). METHODS: Clinical characteristics, biochemical markers detected by tandem mass spectrometry (MS-MS) and gas chromatography-mass spectrometry (GC-MS), and variations of ACAT1 gene of the 8 patients were reviewed. RESULTS: Three patients were diagnosed by newborn screening and were asymptomatic. Five patients showed dyspnea and metabolic acidosis through high risk screening. Blood methylcrotonyl carnitine (C5:1) were 0.43 (0.20-0.89) µmol/L and 3-hydroxyisovaleryl carnitine(C5-OH) were 1.37 (0.98-3.40) µmol/L. Both were significantly higher than those of healthy controls (P<0.01). Urinary 2-methyl-3-hydroxybutyric acid was 56.04 (7.69-182.20) and methylcrotonyl glycine was 42.83 (9.20-127.01), both were higher than normal levels. In 5 patients urinary 2-methyl-3-hydroxybutyric acid level was remarkably decreased (P<0.05) after treatment. Analysis of ACAT1 gene mutation was performed in six families. Missense variations were detected in 78.6% of the cases. 42.8% of the 7 BKD patients have carried c.1124A>G (p.N375S) variant, which accounted for 28.6% of all 14 mutant alleles. Four novel variants, namely c.229delG (p.E77KfsTer10), c.373G>T (p.V125F), c.419T>G (p.L140R) and c.72+1G>A, were discovered. Pathogenicity assessment of two highly conservative missense variants (p.V125F) and (p.L140R) were 0.994 and 1.0 (Scores obtained from PolyPhen2), and PROVEAN scores were -4.652 and -5.399, respectively. c.72+1g>a was suspected (by Human Splicing Finder) to alter the wild type donor motif and most probably affect the splicing. CONCLUSION: Clinicians should consider MS/MS and GC/MS testing for those with unexplained neurological symptoms and metabolic acidosis in order to attain early diagnosis of BKD. Genetic testing should be used to confirm the diagnosis.

Alpha-Methylacetoacetic Aciduria in an Rh-Negative Pregnant Omani Woman With Breech Presentation Delivered With Favourable Outcome.

BACKGROUND: Alpha-methylacetoacetic aciduria, an autosomal recessive disorder of isoleucine and ketone body metabolism, is caused by a mutation in the acetyl coenzyme A acetyltransferase-1 gene (ACAT1; 607809) on chromosome 11q22. Ketoacidotic episodes in such patients are triggered by stress situations with increased energy demands. Pregnancy, surgical procedures, and prolonged fasting are potential triggers for metabolic crisis in such cases. CASE: A young Rh-negative Omani woman with alpha-methylacetoacetic aciduria is described here during her second pregnancy. Her metabolic condition was detected at the age of 18 months. She was successfully delivered of a clinically healthy baby through emergency CS for breech presentation. CONCLUSION: Prompt management by a multidisciplinary team is vital to avoid metabolic crisis and to promote a favourable outcome in these cases.

Recent advances in understanding beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency.

Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency (OMIM #203750, *607809) is an inborn error of metabolism that affects isoleucine catabolism and ketone body metabolism. This disorder is clinically characterized by intermittent ketoacidotic crises under ketogenic stresses. In addition to a previous 26-case series, four series of T2-deficient patients were recently reported from different regions. In these series, most T2-deficient patients developed their first ketoacidotic crises between the ages of 6 months and 3 years. Most patients experienced less than three metabolic crises. Newborn screening (NBS) for T2 deficiency is performed in some countries but some T2-deficient patients have been missed by NBS. Therefore, T2 deficiency should be considered in patients with severe metabolic acidosis, even in regions where NBS for T2 deficiency is performed. Neurological manifestations, especially extrapyramidal manifestations, can occur as sequelae to severe metabolic acidosis; however, this can also occur in patients without any apparent metabolic crisis or before the onset of metabolic crisis.

Clinical and molecular analysis of 6 Chinese patients with isoleucine metabolism defects: identification of 3 novel mutations in the HSD17B10 and ACAT1 gene.

Hydroxysteroid (17ß) dehydrogenase 10 (HSD10) and mitochondrial acetoacetyl-CoA thiolase (ß-KT) are two adjacent enzymes for the degradation of isoleucine, thus HSD10 and ß-KT deficiencies are confusing at an early stage because of nearly the same elevation of typical metabolites in urine, such as 2-methyl-3-hydroxybutyric acid (2M3HBA) and tiglylglycine (TG). In order to better understand the differences between these two disorders, we described the clinical and molecular characteristics of two HSD10 deficiency patients and four ß-KT deficiency patients. ß-KT deficiency patients had a much more favorable outcome than that of HSD10 deficiency patients, indicating that the multifunction of HSD10, especially neurosteroid metabolic activity, other than only enzymatic degradation of isoleucine, is involved in the pathogenesis of HSD10 deficiency. Two different mutations, a novel mutation p.Ile175Met and a reported mutation p.Arg226Gln, were detected in the HSD17B10 gene of HSD10 deficiency patients. Six different mutations, including four known mutations: p.Ala333Pro, p.Thr297Lys, c.83_84delAT, c.1006-1G > C, and two novel mutations: p.Thr277Pro and c.121-3C > G were identified in the ACAT1 gene of ß-KT deficiency patients. In general, DNA diagnosis played an important role in distinguishing between these two disorders.

Clinical presentation and outcome in a series of 32 patients with 2-methylacetoacetyl-coenzyme A thiolase (MAT) deficiency.

2-methylacetoacetyl-coenzyme A thiolase (MAT) deficiency, also known as beta-ketothiolase deficiency, is an inborn error of ketone body utilization and isoleucine catabolism. It is caused by mutations in the ACAT1 gene and may present with metabolic ketoacidosis. In order to obtain a more comprehensive view on this disease, we have collected clinical and biochemical data as well as information on ACAT1 mutations of 32 patients from 12 metabolic centers in five countries. Patients were between 23months and 27years old, more than half of them were offspring of a consanguineous union. 63% of the study participants presented with a metabolic decompensation while most others were identified via newborn screening or family studies. In symptomatic patients, age at manifestation ranged between 5months and 6.8years. Only 7% developed a major mental disability while the vast majority was cognitively normal. More than one third of the identified mutations in ACAT1 are intronic mutations which are expected to disturb splicing. We identified several novel mutations but, in agreement with previous reports, no clear genotype-phenotype correlation could be found. Our study underlines that the prognosis in MAT deficiency is good and MAT deficient individuals may remain asymptomatic, if diagnosed early and preventive measures are applied.

Mitochondrial acetoacetyl-CoA thiolase deficiency: basal ganglia impairment may occur independently of ketoacidosis.

BACKGROUND: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency affects ketone body and isoleucine catabolism. Neurological impairment may occur secondary to ketoacidotic episodes. However, we observed neuromotor abnormalities without ketoacidotic events in two T2-deficient families. We hypothesized that the neurological signs were related to the genetic defect and may occur independently of ketoacidotic episodes. We therefore conducted a retrospective review on a French T2-deficient patient series searching for neuromotor impairment. METHODS: In total, 26 cases were retrospectively analysed for clinical, biological and neuroimaging data. RESULTS: Neurological findings were observed for 6/26 (23%) patients. Among these, two had never experienced ketoacidotic episodes, though they developed extrapyramidal signs with putamen involvement. Two of the other four patients developed neurological abnormalities before the first ketoacidotic crisis, with putamen involvement in one case. The third patient developed extrapyramidal symptoms more than 10 years after the initial decompensation with globus pallidus involvement. The last patient developed extrapyramidal signs immediately after a severe ketoacidotic crisis with putaminal lesions. CONCLUSIONS: Most T2-deficient patients achieved normal neurodevelopment. However, on account of the role of T2 in isoleucine catabolism, these patients are potentially exposed to accumulation of toxic isoleucine-derived metabolites, which may contribute to neurological impairment. Our findings confirm previous observations that neurological symptoms in T2 deficiency may occur unrelated to ketoacidosis. The role of protein restriction as a preventive measure against neurological symptoms could not be established in this study and deserves further evaluation. Long-term follow-up data on children diagnosed by newborn screening may clarify the pathogenesis of this neurometabolic association.

Characterization and outcome of 41 patients with beta-ketothiolase deficiency: 10 years' experience of a medical center in northern Vietnam.

Beta-ketothiolase (T2) deficiency is an inherited disease of isoleucine and ketone body metabolism caused by mutations in the ACAT1 gene. Between 2005 and 2016, a total of 41 patients with T2 deficiency were identified at a medical center in northern Vietnam, with an estimated incidence of one in 190,000 newborns. Most patients manifested ketoacidotic episodes of varying severity between 6 and 18 months of age. Remarkably, 28% of patients showed high blood glucose levels (up to 23.3 mmol/L). Ketoacidotic episodes recurred in 43% of patients. The age of onset, frequency of episodes, and identified genotype did not affect patient outcomes that were generally favorable, with the exception of seven cases (five died and two had neurological sequelae). Custom-tailored acute and follow-up management was critical for a positive clinical outcome. Two null mutations, c.622C>T (p.Arg208*) and c.1006-1G>C (p.Val336fs), accounted for 66% and 19% of all identified ACAT1 mutant alleles, respectively. Most patients showed characteristic biochemical abnormalities. A newborn screening program could be expected to have a high yield in Vietnam. Investigation findings of haplotypes linked to the most common ACAT1 mutation (c.622C>T) are consistent with an ancient common founder of mutation-bearing chromosomes belonging to the Kinh ethnic population. The direct management and long-term follow-up of a large number of T2-deficient patients enabled us to study the natural history of this rare disease.

Beta-ketothiolase deficiency: An unusual cause of recurrent ketoacidosis.

Kiliç-Yildirim G, Durmus-Aydogdu S, Ceylaner S, Sass JO. Beta-ketothiolase deficiency: An unusual cause of recurrent ketoacidosis. Turk J Pediatr 2017; 59: 471-474. Beta-ketothiolase deficiency (mitochondrial acetoacetyl-CoA thiolase, MAT or T2 deficiency) is a rare autosomal recessive disorder of isoleucine and ketone body metabolism due to acetyl-CoA acetyltransferase-1 (ACAT1) gene mutations. The disease is characterized by recurrent episodes of ketoasidosis which starts with vomiting and followed by dehydration and tachypnea. Here, we present a patient who was admitted to the hospital with severe acidosis and dehydration because of vomiting induced by protein rich nutrient and was diagnosed with MAT deficiency. 3-hydroxy-butyric acid, acetoacetic acid and 3-hydroxy-iso-valeric acid levels were significantly increased and tiglyglycine as trace amount in the urine organic acid analysis of the patient. Genetic analysis for ACAT-1 showed compound heterozygosity for the mutations c.949G > A (p.D317N) and c.951C > T (p.D317D), which both are known to cause exon 10 skipping and to be pathogenic missense mutations.

Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site.

Beta-ketothiolase deficiency, also known as mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, is an autosomal recessive disease caused by mutations in the acetyl­CoA acetyltransferase 1 (ACAT1) gene. A German T2­deficient patient that developed a severe ketoacidotic episode at the age of 11 months, was revealed to be a compound heterozygote of a previously reported null mutation, c.472A>G (p.N158D) and a novel mutation, c.949G>A (p.D317N), in ACAT1. The c.949G>A mutation was suspected to cause aberrant splicing as it is located within an exonic splicing enhancer sequence (c. 947CTGACGC) that is a potential binding site for serine/arginine­rich splicing factor 1. A mutation in this sequence, c.951C>T, results in exon 10 skipping. A minigene construct was synthesized that included exon 9­truncated intron 9­exon 10­truncated intron 10­exon 11, and the splicing of this minigene revealed that the c.949G>A mutant construct caused exon 10 skipping in a proportion of the transcripts. Furthermore, additional substitution of G for C at the first nucleotide of exon 10 (c.941G>C) abolished the effect of the c.949G>A mutation. Transient expression analysis of the c.949G>A mutant cDNA revealed no residual T2 activity in the mutated D317N enzyme. Therefore, c.949G>A (D317N) is a pathogenic missense mutation, and diminishes the effect of an exonic splicing enhancer and causes exon 10 skipping. The present study demonstrates that a missense mutation, or even a synonymous substitution, may disrupt enzyme function by interference with splicing.

[Analysis of clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency].

OBJECTIVE: To investigate the clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency (BKTD). METHODS: Clinical features and laboratory test data were collected. The probands were monozygotic twin brothers. Genomic DNA was isolated from peripheral blood leukocytes obtained from the probands and their family members. Molecular genetic testing of the ACAT1 gene was carried out. RESULTS: The probands have presented with fever, vomiting and severe ketoacidosis. By arterial blood gas testing, pH was determined to be 7.164, bicarbonate was 4.0 mmol/L, and urine ketone was ++++. Urinary organic acid gas chromatography-mass spectrometry analysis showed excessive excretion of 3-hydroxybutyric acid, 2-methyl-3-hydroxybutyric acid and tiglylglycine. Increased 3-hydroxybutyrylcarnitine (C4-OH), tiglylcarnitine(C5:1) and 3-hydroxyisovalerylcarnitine (C5-OH) levels. The clinical phenotype of proband's parents were both normal, but an elder sister turned out to be an affected patient. Genetic analysis has identified two heterozygous mutations [c.622C>T(p.R208X) and c.653C>T (p.S218F)] in the proband, which were respectively detected in the mother and father. The c.653C>T (p.S218F) mutation was not found among the 100 healthy controls and has not been included in the Human Gene Mutation Database(HGMD). CONCLUSION: The primary clinical manifestations of BKTD is ketoacidosis. Urine organic acid and blood acylcarnitine analyses play an important role in the diagnosis of the disease. The compound heterozygous of ACAT1 gene mutations probably underlie the BKTD in our patient.