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.

Molecular basis of two-exon skipping (exons 12 and 13) by c.1248+5g>a in OXCT1 gene: study on intermediates of OXCT1 transcripts in fibroblasts.

The molecular basis of simultaneous two-exon skipping induced by a splice-site mutation has yet to be completely explained. The splice donor site mutation c.1248+5g>a (IVS13) of the OXCT1 gene resulted predominantly in skipping of exons 12 and 13 in fibroblasts from a patient (GS23) with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency. We compared heteronuclear RNA (hnRNA) intermediates between controls' and GS23's fibroblasts. Our strategy was to use RT-PCR of hnRNA to detect the presence or absence of spliced exon clusters in RNA intermediates (SECRIs) comprising sequential exons. Our initial hypothesis was that a SECRI comprising exons 12 and 13 was formed first followed by skipping of this SECRI in GS23 cells. However, such a pathway was revealed to be not a major one. Hence, we compared the intron removal of SCOT transcript between controls and GS23. In controls, intron 11 was the last intron to be spliced and the removal of intron 12 was also rather slow and occurred after the removal of intron 13 in a major pathway. However, the mutation in GS23 cells resulted in retention of intron 13, thus causing the retention of introns 12 and 11. This "splicing paralysis" may be solved by skipping the whole intron 11-exon 12-intron 12-exon 13-mutated intron 13, resulting in skipping of exons 12 and 13.

A neonatal-onset succinyl-CoA:3-ketoacid CoA transferase (SCOT)-deficient patient with T435N and c.658-666dupAACGTGATT p.N220_I222dup mutations in the OXCT1 gene.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency causes episodic ketoacidotic crises and no apparent symptoms between them. Here, we report a Japanese case of neonatal-onset SCOT deficiency. The male patient presented a severe ketoacidotic crisis, with blood pH of 7.072 and bicarbonate of 5.8 mmol/L at the age of 2 days and was successfully treated with intravenous infusion of glucose and sodium bicarbonate. He was diagnosed as SCOT deficient by enzymatic assay and mutation analysis. At the age of 7 months, he developed a second ketoacidotic crisis, with blood pH of 7.059, bicarbonate of 5.4 mmol/L, and total ketone bodies of 29.1 mmol/L. He experienced two milder ketoacidotic crises at the ages of 1 year and 7 months and 3 years and 7 months. His urinary ketone bodies usually range from negative to 1+ but sometimes show 3+ (ketostix) without any symptoms. Hence, this patient does not show permanent ketonuria, which is characteristic of typical SCOT-deficient patients. He is a compound heterozygote of c.1304C > A (T435N) and c.658-666dupAACGTGATT p.N220_I222dup. mutations in the OXCT1 gene. The T435N mutation was previously reported as one which retained significant residual activity. The latter novel mutation was revealed to retain no residual activity by transient expression analysis. Both T435N and N220_I222 lie close to the SCOT dimerization interface and are not directly connected to the active site in the tertiary structure of a human SCOT dimer. In transient expression analysis, no apparent interallelic complementation or dominant negative effects were observed. Significant residual activity from the T435N mutant allele may prevent the patient from developing permanent ketonuria.

De-phosphorylation of TRalpha-1 by p44/42 MAPK inhibition enhances T(3)-mediated GLUT5 gene expression in the intestinal cell line Caco-2 cells.

Thyroid hormone and p44/42 MAPK inactivation are important in intestinal differentiation. We demonstrated not only that treatment with p44/42 MAPK inhibitor U0126 in intestinal cell line Caco-2 cells reduced the phosphorylation of serine and threonine residues of TRalpha-1, but also that T(3) and U0126 synergistically induced GLUT5 gene expression. EMSA demonstrated that the binding activity of TRalpha-1-RXR heterodimer on GLUT5-TRE in nuclear proteins of Caco-2 cells was synergistically enhanced by co-incubation in vitro with T(3) and CIAP, which strongly de-phosphorylates proteins. ChIP and transfection assays revealed that co-treatment of T(3) and U0126 induces TRalpha-1-RXR binding to GLUT5-TRE on the human GLUT5 enhancer region, and recruitment of the transcriptional complex in cells. These results suggest that inactivation of p44/42 MAPK enhances T(3)-induced GLUT5 gene expression in Caco-2 cells through increasing TRalpha-1 transactivity and binding activity to the GLUT5-TRE, probably due to de-phosphorylation of TRalpha-1.

Triiodothyronine (T3) and fructose coordinately enhance expression of the GLUT5 gene in the small intestine of rats during weaning period.

Jejunal GLUT5 is elevated with triiodothyronine (T(3)) during weaning of rats. A perfusion of fructose into the small intestine of T(3)-injected rats at 21 d induced expression of the GLUT5 gene, but one into that of vehicle-injected rats did not. These results suggest that T(3) and fructose coordinately enhance jejunal expression of the GLUT5 gene in rats during weaning period.

The critical period for thyroid hormone responsiveness through thyroid hormone receptor isoform alpha in the postnatal small intestine.

During second and third weeks after birth in rats, serum thyroid hormone level is elevated. In this study, we investigated the jejunal expression of thyroid hormone receptor (TR) alpha in developing rats. The TRalpha-1 mRNA level and TRalpha-1/TRalpha-2 mRNA ratio increased two-fold from 5 to 13 days after birth. This high level of TRalpha-1 mRNA was maintained until 20 days and then decreased to the basal level by the end of weaning period at 27 days; however, the level of TRalpha-2 mRNA remained unchanged throughout the developmental period. The increase in the TRalpha-1/TRalpha-2 mRNA ratio from 5 to 13 days was accompanied by an initial rise in the levels of mRNA for hexose transporters in the jejunum. Administration of T(3) during the suckling period (8-13 days) caused a 50% increase in the TRalpha-1/TRalpha-2 mRNA ratio, while administration of T(3) on days 12-17 and days 16-21, but not on days 22-27, caused a two to four-fold increase in the levels of mRNA for hexose transporters. These results suggest that a transient variation in the TRalpha-1/TRalpha-2 expression ratio is closely related to the critical period of thyroid hormone responsiveness for hexose transporters expression in the developing rat jejunum.

Evaluation of oxidative stress during apoptosis and necrosis caused by D-galactosamine in rat liver.

Eighteen and twenty-four hours after intraperitoneal administration of D-galactosamine (1g/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared with that in the control group given saline. Histological examinations including the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method found apoptotic hepatocytes 18 hr after the administration of D-galactosamine. Caspase-3 activity was barely detectable in the plasma of control rats, but increased significantly 24 hr after drug administration along with a dramatic increase in glutamate-oxaloacetate transaminase (GOT). These results indicated that D-galactosamine causes apoptosis in the liver by activating caspase-3, which is released to the plasma by secondary necrosis. The concentration of lipid hydroperoxides in the liver increased significantly 24 hr after D-galactosamine administration. In contrast, the concentration of vitamin C in the liver decreased significantly 18 and 24 hr after D-galactosamine administration. These results suggest that D-galactosamine induces severe oxidative stress in the liver, leading to extensive necrosis.

Major intestinal coactivator p300 strongly activates peroxisome proliferator-activated receptor in intestinal cell line, Caco-2.

We have previously reported that several genes related to intestinal fatty acid and vitamin A metabolism are coordinately regulated by peroxisome proliferator-activated receptor (PPAR) [Arch. Biochem. Biophys. 389 (2001) 41; Biochim. Biophys. Acta 1531 (2001) 68]. In this study, we demonstrated that PPAR alpha and PPAR delta interacted with endogenous coactivators in intestinal cell line, Caco-2 in a ligand specific manner. We isolated rat cDNA clones encoding the nuclear receptor interaction domains of the two transcriptional coactivators, CREB-binding protein (CBP) and p300. Expression level of CBP mRNA was relatively low in the small intestine, while p300 mRNA was ubiquitously expressed in various tissues including the small intestine in the rat. Southern blot analysis revealed that these coactivators were encoded by different genes. Mammalian two-hybrid assays in Caco-2 cells revealed that p300 interacted with PPAR alpha or PPAR delta in the presence of their specific ligands more efficiently than CBP did. These results suggest that the major intestinal coactivator, p300 strongly interacts with PPAR alpha and PPAR delta.