Practical assay method of cytosolic acetoacetyl-CoA thiolase by rapid release of cytosolic enzymes from cultured lymphocytes using digitonin.

We designed a simple approach to determine cytosolic acetoacetyl-CoA thiolase (CT) activity for differential diagnosis of ketone body catabolic defects, using rapid cell-subfractionation of cultured lymphocytes with digitonin. Efficiency of cell subfractionation was determined by measurement of lactate dehydrogenase and citrate synthetase as marker enzymes for cytosol and organelle fractions, respectively, and confirmed by immunotitration and immunoblotting using antibodies against cytosolic and mitochondrial thiolases, respectively. In the condition of best separation taken in the presence of 1 mg/ml digitonin, acetoacetyl-CoA thiolase activities in the presence of K+ ion in the cytosol and organelle fractions were 138.3+/-39.2 and 84.0+/-16.2 nmol/min/ml, respectively. The thiolase activity in the organelle fraction was doubled by the presence of K+ ion, whereas that in the cytosol fraction was not affected. The thiolase activity in the organelle fraction was reduced by the treatment of anti-mitochondrial acetoacetyl-CoA thiolase (T2) antibody but not by anti-CT antibody. On the other hand, that in the cytosol fraction was significantly decreased by anti-CT antibody but not by anti-T2 antibody. These data suggested that T2 was collected in the organelle fraction, and that CT activity could be assessed by measurement of the thiolase activity in the cytosolic fraction. Succinyl-CoA: 3-ketoacid CoA transferase (SCOT), whose defect is the third inherited disorder of ketone body catabolism, was collected in the organelle fraction. Hence, this method will prove to be useful for accurate assessment of defects of CT as well as T2 or SCOT, all involved in ketone body catabolism.

Hemispheric asymmetry of event-related potentials in a patient with callosal disconnection syndrome: a comparison of auditory, visual and somatosensory modalities.

To investigate whether callosal lesions affect the distribution of event-related potentials (ERP) between the two hemispheres and whether hemispheric ERP distribution differs among sensory modalities, a patient with interhemispheric disconnection syndrome and 47 controls were subjected to an oddball paradigm. High (target) and low tone bursts for auditory, red (target) and green lights for visual and electrical stimuli delivered to the index (target) or fifth finger for somatosensory ERPs were presented to the unilateral ear, visual field and hand, respectively. The subjects were instructed to press a button with the hand on the stimulated side. The results showed that the hemispheric asymmetry of the patient's auditory ERPs was not significantly different from that of the controls, regardless of which ear was stimulated. In contrast, the visual and somatosensory ERPs showed a delay of the P3 latency and an attenuation of the N1-P2 and N2-P3 amplitude over the hemisphere ipsilateral to the stimulus, regardless of the stimulated side. These findings suggest that the source of P3 generation is relatively lateralized to the hemisphere contralateral to the stimulus, and that the callosal transfer of visual and somatosensory information is involved in the P3 generation in the hemisphere ipsilateral to the stimulus.

Prenatal diagnosis in a family with mitochondrial acetoacetyl-coenzyme A thiolase deficiency with the use of the polymerase chain reaction followed by the heteroduplex detection method.

Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency is an organic aciduria which affects isoleucine and ketone body catabolism. GK16 (the index patient) was affected with this disorder and previous studies had revealed that GK16 was a compound heterozygote with IVS8(+1) gt to tt and A301P mutations. In a subsequent pregnancy, prenatal diagnosis was performed and the fetus's amniocytes were analysed by the polymerase chain reaction (PCR) followed by the heteroduplex detection method on a Mutation Detection Enhancement gel. The fetus was identified as a carrier of the IVS8(+1) mutation. We confirmed the diagnosis by immunoblot analysis of extracted amniocytes and gene analysis with blood filter paper after delivery. This is the first report of prenatal diagnosis of this disorder at the gene level.

Molecular, biochemical, and clinical characterization of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in two further patients.

The molecular basis of mitochondrial acetoacetyl-CoA thiolase (T2) deficiency was studied in two patients (GK11 and GK16). Fibroblasts from each patient had detectable immunoreactive T2 polypeptide (CRM). In pulse-chase experiments, fibroblasts from GK11 had two types of CRM: one (type I CRM) disappeared after a 24-hr chase and migrated more slowly than that of the normal control; the other (type II CRM) was detected with a small amount even after a 72-hr chase and had normal electrophoretic mobility. GK16's fibroblasts had a CRM (type III) which was also detectable even after a 72-hr chase and showed a slower mobility than type I CRM. By analyzing amplified cDNA and genomic fragments, we showed that both patients are genetic compounds; GK11 for the mutations N158D and T297M, and GK16 for the mutations A301P and IVS8 (+1). Expression analyses confirmed that mutant T2 subunits with N158D, T297M, and A301P correspond to type I, II, and III CRM, respectively. Among them, only the mutant T2 polypeptide with T297M appeared to have a detectable residual activity, in spite of its instability. Cotransfection of two cDNAs containing N158D and T297M suggested that heterotetramer formation reduces residual activity in GK11 cells.

Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation.

Interstitial deletion of the long arm of chromosome 11 determined by fluorescence in situ hybridization.

An interstitial deletion, del(11)(q14q22), found in a female infant was examined by fluorescence in situ hybridization with cosmid DNA markers mapped on the long arm of chromosome 11. Three cosmids mapped on 11q14.1-11q22.1 region were not hybridized to the del(11) chromosome, while all the other DNA markers mapped on 11cen-11q14.1 and 11q23.1-11qter region gave hybridization signals on the del(11) chromosome. Cytogenetic analysis after R-banding confirmed an apparent deletion of 11q14-q22, but containing a small R-negative band, a part of 11q22.3 and/or 11q14.1, in the middle part of del(11) chromosome. The karyotype thus was determined to be 46,XX,del(11)(q14.1q22.3).

Biochemical and immunochemical study of seven families with 3-ketothiolase deficiency: diagnosis of heterozygotes using immunochemical determination of the ratio of mitochondrial acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase proteins.

The possibility of identifying heterozygotes of 3-ketothiolase deficiency, an inborn error of metabolism caused by a defect of mitochondrial acetoacetyl-CoA thiolase (T2), was tested in seven unrelated families by using enzymatic assay of thiolase activity and immunoblot analysis. The ratio of acetoacetyl-CoA thiolase activities, in the presence and absence of K+ ion (+K/-K ratio), in fibroblasts from 15 normal controls was around 2.0 (1.8 to 2.4), whereas the +K/-K ratio in eight patients was always 1.0. The ratio for the 13 obligate carriers ranged from 1.4 to 1.9, causing a minor overlap with control. Identification of heterozygote cells by immunoblot analysis, using anti-T2 antibody alone as a probe, was difficult, as previously reported. We therefore carried out immunoblot analysis, using as probes a mixture of anti-T2 antibody and the antibody against mitochondrial 3-ketoacyl-CoA thiolase (T1), another mitochondrial thiolase, and determined the ratio of the intensities of the T2 and T1 bands (T2/T1 ratio) using a densitometer. When the T2/T1 ratio was calculated, there was no overlap between the heterozygotes and normal controls. Hence, the heterozygotes can be unambiguously identified using this method. The thiolase activities and T2/T1 proteins in immunoblotting were detectable in peripheral lymphocytes, rectal mucosa, amniocytes, and liver. Thus, the postnatal diagnosis of 3-ketothiolase deficiency can be readily made using lymphocytes or rectal mucosa. The applicability of these methods in amniocytes indicates that prenatal diagnosis of this disease should be possible.

Molecular studies of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in the two original families.

We describe mutations identified in stored skin fibroblast cell lines from two original probands (JB and JM), first reported with 2-methylacetoacetic aciduria, and shown later to have a deficiency of the K(+)-activated enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2). JB is homozygous for a 4-base insertion (GCAG) which is derived mutation. The primary mutation is an AG/gt to AG/gc transition at the 5'-splice-junction site in intron 11. An alternative splice site 4 bp downstream (Ggcag/gt) is used which causes a frame shift and replaces 39 C-terminal residues by 70 nonfunctional residues. JM is homozygous for a mutation in the translation-initiation codon (ATG to AAG). By expression analyses the JB mutation (IVS11nt2) causes an unstable T2 polypeptide and the JM mutation (M1K) severely impairs T2 mRNA translation. The JB allele associates with Dutch ancestry (no consanguinity) and the JM allele with Chilean ancestry (distant consanguinity).

Interstitial deletion of the long arm of chromosome 11: report of a case and review of the literature.

A 12-month-old female infant with developmental delay, growth retardation, and dysmorphic features including dolichocephaly, telecanthus, ptosis, flat nasal bridge, anteverted nares, high-arched palate, carp-shaped mouth, micro-retrognathia, and low-set and posteriorly rotated ears was found to have an interstitial deletion of chromosome 11 involving bands q14-q22. Immunoblot analysis of her fibroblasts revealed a normal amount of mitochondrial acetoacetyl-coenzyme A thiolase, of which gene locus has been assigned to chromosome 11q22.3-q23.1. This result suggested that the region around the boundary of 11q22.3-q23.1 was intact in this patient.

Further analysis of mutant thiolase protein in fibroblasts from a Japanese boy with 3-ketothiolase deficiency.

We examined the mutant protein of mitochondrial acetoacetyl-CoA thiolase (mutant T2) in fibroblasts from a Japanese boy with 3-ketothiolase deficiency. The molecular size of the mutant T2 protein, determined by pulse labeling and SDS/PAGE, was intermediate between the mature subunit and the precursor of T2. To characterize the mutant T2 protein, pulse-labeling and rhodamine 6G inhibition of mitochondrial transport in fibroblasts, cell-free translation experiments, and family studies by thiolase assay, immunoblotting, and pulse-labeling were carried out. The mutant T2 was detectable as early as a 10-min pulse. The probable precursor of the mutant T2 was not detectable in either the rhodamine 6G inhibition or cell-free translation experiments. In the parents, the K+ ion dependency of acetoacetyl-CoA thiolase activity was low and the T2 bands in immunoblots were faint. It would thus appear that the parents are heterozygotes of this disease. In pulse-labeling, only a band for the mutant T2 was detected in the patient and a single band for the normal mature subunit of T2 in the father; both bands were detected in the mother. These findings suggested that the mutant T2 in the patient was inherited from the mother, and that the expression of another mutant allele of the father may be either abolished or scanty.