Yuka Aoyama

Ketone body metabolism and its defects.

Acetoacetate (AcAc) and 3-hydroxybutyrate (3HB), the two main ketone bodies of humans, are important vectors of energy transport from the liver to extrahepatic tissues, especially during fasting, when glucose supply is low. Blood total ketone body (TKB) levels should be evaluated in the context of clinical history, such as fasting time and ketogenic stresses. Blood TKB should also be evaluated in parallel with blood glucose and free fatty acids (FFA). The FFA/TKB ratio is especially useful for evaluation of ketone body metabolism. Defects in ketogenesis include mitochondrial HMG-CoA synthase (mHS) deficiency and HMG-CoA lyase (HL) deficiency. mHS deficiency should be considered in non-ketotic hypoglycemia if a fatty acid beta-oxidation defect is suspected, but cannot be confirmed. Patients with HL deficiency can develop hypoglycemic crises and neurological symptoms even in adolescents and adults. Succinyl-CoA-3-oxoacid CoA transferase (SCOT) deficiency and beta-ketothiolase (T2) deficiency are two defects in ketolysis. Permanent ketosis is pathognomonic for SCOT deficiency. However, patients with “mild” SCOT mutations may have nonketotic periods. T2-deficient patients with “mild” mutations may have normal blood acylcarnitine profiles even in ketoacidotic crises. T2 deficient patients cannot be detected in a reliable manner by newborn screening using acylcarnitines. We review recent data on clinical presentation, metabolite profiles and the course of these diseases in adults, including in pregnancy.

The first case in Asia of 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (HSD10 disease) with atypical presentation

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (2M3HBD) deficiency (HSD10 disease) is a rare inborn error of metabolism, and <30 cases have been reported worldwide. This disorder is typically characterized by progressive neurodegenerative disease from 6 to 18 months of age. Here, we report the first patient with this disorder in Asia, with atypical clinical presentation. A 6-year-old boy, who had been well, presented with severe ketoacidosis following a 5-day history of gastroenteritis. Urinary organic acid analysis showed elevated excretion of 2-methyl-3-hydroxybutyrate and tiglylglycine. He was tentatively diagnosed with β-ketothiolase (T2) deficiency. However, repeated enzyme assays using lymphocytes showed normal T2 activity and no T2 mutation was found. Instead, a hemizygous c.460G>A (p.A154T) mutation was identified in the HSD17B10 gene. This mutation was not found in 258 alleles from Japanese subjects (controls). A normal level of the HSD17B10 protein was found by immunoblot analysis but no 2M3HBD enzyme activity was detected in enzyme assays using the patient’s fibroblasts. These data confirmed that this patient was affected with HSD10 disease. He has had no neurological regression until now. His fibroblasts showed punctate and fragmented mitochondrial organization by MitoTracker staining and had relatively low respiratory chain complex IV activity to those of other complexes.

Carnitine–acylcarnitine translocase deficiency: Two neonatal cases with common splicing mutation and in vitro bezafibrate response

BACKGROUND Mitochondrial fatty acid oxidation (FAO) disorders are among the causes of acute encephalopathy- or myopathy-like illness. Carnitine-acylcarnitine translocase (CACT) deficiency is a rare FAO disorder, which represent an energy production insufficiency during prolonged fasting, febrile illness, or increased muscular activity. CACT deficiency is caused by mutations of the SLC25A20 gene. Most patients developed severe metabolic decompensation in the neonatal period and died in infancy despite aggressive treatment. PATIENTS AND METHODS We herein report the clinical findings of two unrelated cases of CACT deficiency with mutation confirmation, and in vitro bezafibrate responses using in vitro probe acylcarnitine (IVP) assay. Patients 1 and 2 are products of nonconsanguineous parents. Both patients developed cardiac arrest at day 3 of life but survived the initial events. Their blood chemistry revealed hypoglycemia and metabolic acidosis. The acylcarnitine profiles in both patients demonstrated increased long-chain acylcarnitines, suggesting CACT or carnitine palmitoyltransferase-2 (CPT2) deficiency. RESULTS The mutation analysis identified homozygous IVS2-10T>G in the SLC25A20 gene in both patients, confirming the diagnosis of CACT deficiency. The IVP assay revealed increased C16, C16:1, but decreased C2 with improvement by bezafibrate in the cultured fibroblasts. The short-term clinical trial of bezafibrate in Patient 1 did not show clinical improvement, and died after starting the trial for 6 months. CONCLUSION This splicing mutation has been identified in other Asian populations indicating a possible founder effect. IVP assay of cultured fibroblasts could determine a response to bezafibrate treatment. A long-term clinical trial of more enrolled patients is required for evaluation of this therapy.

Beta-Ketothiolase Deficiency Resolving Challenges in Diagnosis

Beta-ketothiolase deficiency is an inherited disorder of ketone body metabolism and isoleucine catabolism. It typically manifests as recurrent ketoacidotic episodes with characteristic abnormalitie...

Application of multiplex ligation-dependent probe amplification, and identification of a heterozygous Alu-associated deletion and a uniparental disomy of chromosome 1 in two patients with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) deficiency is an autosomal recessive disorder affecting the leucine catabolic pathway and ketone body synthesis, and is clinically characterized by metabolic crises with hypoketotic hypoglycemia, metabolic acidosis and hyperammonemia. In the present study, we initially used PCR with genomic followed by direct sequencing to investigate the molecular genetic basis of HMGCL deficiency in two patients clinically diagnosed with the condition. Although we identified a mutation in each patient, the inheritance patterns of these mutations were not consistent with disease causation. Therefore, we investigated HMGCL using multiplex ligation-dependent probe amplification (MLPA) to determine the copy numbers of all exons. A heterozygous deletion that included exons 2–4 was identified in one of the patients. MLPA revealed that the other patient had two copies for all HMGCL exons. Paternal uniparental isodisomy of chromosome 1 was confirmed in this patient by microarray analysis. These findings indicate that MLPA is useful for the identification of genomic aberrations and mutations other than small-scale nucleotide alterations. To the best of our knowledge, this is the first study describing HMGCL deficiency caused by uniparental disomy.

Metabolic encephalopathy in beta-ketothiolase deficiency: The first report from India

Beta-ketothiolase deficiency, or mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, is a rare autosomal recessive disorder affecting isoleucine catabolism and ketone body metabolism. A patient from South India presented with acute ketoacidosis at 11 months of age. During the acute crisis the C5OH (2-methyl-3-hydroxybutyryl) carnitine and C5:1 (tiglyl) carnitine were elevated and large amounts of 2-methyl-3-hydroxybutyrate, tiglylglycine, and 2-methylacetoacetate were excreted. Brain CT showed bilateral basal ganglia lesions. Potassium ion-activated acetoacetyl-CoA thiolase activity was deficient in the patients fibroblasts. The patient is a homozygote for a novel c.578T>G (M193R) mutation. This is the first report of T2 deficiency confirmed by enzyme and molecular analysis from India.

Development of MLPA for human ACAT1 gene and identification of a heterozygous Alu-mediated deletion of exons 3 and 4 in a patient with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency

Mitochondrial acetoacetyl-CoA thiolase deficiency is an autosomal recessive disorder, characterized by intermittent ketoacidosis. We developed a multiplex ligation-dependent probe amplification method for mutation detection in the ACAT1 gene, which encodes this enzyme, and validated it using DNAs from two previously reported patients having partial deletion and duplication in this gene. Using this method, we identified a heterozygous deletion including exons 3-4 in a third patient, likely due to Alu-mediated non-equal homologous recombination between Alu sequences.

Mechanism of paclitaxel resistance in a human prostate cancer cell line, PC3-PR, and its sensitization by cabazitaxel

Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patients prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of βIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.

Single-nucleotide substitution T to A in the polypyrimidine stretch at the splice acceptor site of intron 9 causes exon 10 skipping in the ACAT1 gene

β‐ketothiolase (T2, gene symbol ACAT1) deficiency is an autosomal recessive disorder, affecting isoleucine and ketone body metabolism. We encountered a patient (GK03) with T2 deficiency whose T2 mRNA level was <10% of the control, but in whom a previous routine cDNA analysis had failed to find any mutations. Genomic PCR‐direct sequencing showed homozygosity for c.941‐9T>A in the polypyrimidine stretch at the splice acceptor site of intron 9 of ACAT1. Initially, we regarded this variant as not being disease‐causing by a method of predicting the effect of splicing using in silico tools. However, based on other findings of exon 10 splicing, we eventually hypothesized that this mutation causes exon 10 skipping.

Clinical and Mutational Characterizations of Ten Indian Patients with Beta-Ketothiolase Deficiency

Beta-ketothiolase deficiency (mitochondrial acetoacetyl-CoA thiolase (T2) deficiency) is an inherited disease of isoleucine catabolism and ketone body utilization caused by ACAT1 mutations. We identified ten Indian patients who manifested with ketoacidotic episodes of variable severity. The patients showed increased urinary excretion of isoleucine-catabolic intermediates: 2-methyl-3-hydroxybutyrate, 2-methylacetoacetate, and tiglylglycine. Six patients had a favorable outcome, one died, and three developed neurodevelopmental sequela. Mutational analysis revealed a common (p.Met193Arg) and four novel (p.Ile323Thr, p.Ala215Asn, c.1012_1015dup, and c.730+1G>A) ACAT1 mutations. Transient expression analyses of wild-type and mutant cDNA were performed at 30, 37, and 40°C. A p.Ile323Thr mutant T2 was detected with relative enzyme activity and protein amount of 20% and 25%, respectively, compared with wild type at 37°C; it was more prevalent at 30°C but ablated at 40°C. These findings showed that p.Ile323Thr had a significant residual T2 activity with temperature-sensitive instability. Neither residual enzymatic activity nor mutant T2 protein was identified in p.Met193Arg, p.Ala215Asn, and c.1012_1015dup mutations using supernatants; however, these mutant T2 proteins were detected in insoluble pellets by immunoblot analysis. Expression analyses confirmed pathogenicity of these mutations. T2 deficiency has a likely high incidence in India and p.Met193Arg may be a common mutation in the Indian population.