DBT Genes Research Articles

Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease

SUMMARYAnalysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD.

Identification of two novel BCKDHA mutations in a Chinese patient with maple syrup urine disease

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder affecting branched-chain amino acids. Mutations in the BCKDHA, BCKDHB, and DBT gene impair the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex, resulting in the accumulation of branched-chain amino acids and branched-chain alpha-ketoacid in tissues and plasma. This leads to mental and physical retardation, feeding problems, and a maple syrup odor in the urine. In this study, we describe the clinical and biochemical manifestations of a sporadic mutation in a neonate with classic MSUD. Analysis of the BCKDHA gene revealed a compound heterozygous mutation consisting of two novel missense mutations (p.L103P and p.R265P). Viewing the protein with PyMOL indicated that the p.L103P and p.R265P mutations were, respectively, located in the helical region and core domains of the BCKD's Ela component. The p.L103P mutation affected the hydrophobic cores and is predicted to shorten the helix; the p.R265P mutation can predictably affect the cofactor binding site by ligating the associated manganese ion. In conclusion, we identified two novel missense mutations in the BCKDHA gene in a Chinese patient with MSUD.

Four novel mutations identified in Norwegian patients result in intermittent maple syrup urine disease when combined with the R301C mutation

Maple syrup urine disease (MSUD) is caused by a defect in branched chain α-ketoacid dehydrogenase complex (BCKD), an essential metabolon for the catabolism of the branched chain amino acids. Here, we report four novel mutations in the DBT gene, encoding the transacylase subunit (E2) of BCKD, resulting in intermittent MSUD in seven Norwegian patients. The patients had episodes with neurological symptoms including lethargy and/or ataxia during childhood infections. All seven patients were heterozygous for the annotated R301C mutation. The second allelic mutations were identified in five patients; one nonsense mutation (G62X), two missense mutations (W84C and R376C) and a mutation in the 3′ untranslated region (UTR; c.∗358A>C) in two patients. These four novel mutations result in near depletion of E2 protein, and the common R301C protein contributes predominantly to the residual (14%) cellular BCKD activity. Structural analyses of the mutations implied that the W84C and R376C mutations affect stability of intramolecular domains in E2, while the R301C mutation likely disturbs E2 trimer assembly as previously reported. The UTR mutated allele coincided with a strong reduction in mRNA levels, as did the non-R301C specific allele in two patients where the second mutation could not be identified. In summary, the pathogenic effect of the novel mutations is depletion of cellular protein, and the intermittent form of MSUD appears to be attributed to the residual R301C mutant protein in these patients.

Prenatal diagnosis of a novel mutation, c.529C>T (p.Q177X), in the BCKDHA gene in a family with maple syrup urine disease

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder caused by defective activity of the branched-chain alpha-keto-acid dehydrogenase (BCKD) complex. The disease-causing mutations can affect the BCKDHA, BCKDHB or DBT genes encoding for the E1a, E1b, and E2 subunits, respectively, of the BCKD complex. Here we report a girl who first presented to our clinic at 4 years of age with profound mental retardation. A diagnosis of MSUD was subsequently made based on the results of plasma amino acid analysis. Mutation analysis confirmed that she was homozygous for a novel mutation, c.529C>T (p.Q177X) in BCKDHA, while both parents, who were first cousins, were heterozygous. This enabled us to give an option of prenatal diagnosis to the parents. The prenatal testing for MSUD was performed during the mother's subsequent pregnancy and revealed that the fetus was heterozygous for the mutation. The healthy male neonate was born and his genotype was tested by restriction enzyme analysis, which confirmed the result of the prenatal testing. In summary, a late diagnosis of MSUD in patients without an unusual odour could occur especially in countries without neonatal screening programs as seen in the index patient. Mutation detection was, however, still beneficial to the family since prenatal testing could be performed in subsequent pregnancies. In addition, a novel mutation was found, expanding the mutation spectrum of this disease.

Maple syrup urine disease due to a new large deletion at BCKDHA caused by non‐homologous recombination

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acid (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). Many MSUD-causing mutations have already been described in genes that encode the complex (BCKDHA, BCKDHB and DBT), but up to now only four large deletions are known, all located in the DBT gene. In a previous study we identified a Portuguese MSUD patient with a homozygous deletion of exons 2, 3 and 4 at the BCKDHA gene; however, the corresponding breakpoints and, consequently, the exact deletion extension were not identified. Here, using long-range PCR and sequencing methodologies we were able to refine the characterization of this gross rearrangement. A genomic DNA loss of about 13.8 kb was detected, starting at intron 1 and ending at intron 4, thus encompassing exons 2, 3 and 4. Molecular characterization showed that the deletion junction contained a short sequence whose motif was CGGG. Since this motif is present in introns 1 and 4 of normal genomic DNA, we have hypothesized that non-homologous recombination was the mechanism underlying the identified large deletion, within which the CGGG could be derived either from intron 1 or from intron 4.

Description of the mutations in 15 subjects with variant forms of maple syrup urine disease

In maple syrup urine disease (MSUD), disease-causing mutations can affect the BCKDHA, BCKDHB or DBT genes encoding for the E1 alpha, E1 beta and E2 subunits of the multienzyme branched-chain 2-keto acid dehydrogenase (BCKD) complex. The aim of this study was to screen DNA samples of 15 subjects with distinct well-characterized variant MSUD phenotypes for mutations in the three genes in order to demonstrate a potential correlation between specific nucleotide changes and particular variant phenotypes. The exonic coding sequences of all three genes were studied using genomic DNA and cellular RNA derived from peripheral blood leukocytes. In 37% of the cases (total 30 alleles), disease-causing mutations were located in the BCKDHA, in 46% in the BCKDHB, and in 13% in the DBT gene. Novel mutations occurring homozygously were p.Ala328Thr in the BCKDHA gene and p.Gly249_Lys257del in the DBT gene. Both are associated with a mild MSUD variant. The same holds true for the novel mutations p.Pro200Ala in BCKDHB and p.Phe307Ser in DBT which were identified in heterozygous fashion. Among the known mutant alleles, p.Gly278Ser in the BCKDHB gene was relatively frequent and also associated with a mild MSUD variant. The results of this study indicate that genotyping may be predictive of clinical severity of variant MSUD phenotypes and might be of prognostic value particularly in subjects with variant MSUD identified in newborn screening in whom early treatment fortunately slows the natural course of the disease.

Mutational spectrum of maple syrup urine disease in Spain

Mutations in any of the three different genes BCKDHA, BCKDHB, and DBT encoding for the E1alpha, E1beta, and E2 catalytic components of the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex can cause maple syrup urine disease (MSUD). The disease presents heterogeneous clinical and molecular phenotypes. Severity of the disease ranges from the classical to the mildest variant types. Here, we describe the MSUD genotypes and related phenotypes in a cohort of 33 Spanish patients. Based on complementation testing, we selected 15 patients as defective in E1beta, 10 in E1alpha, seven in E2l; one remains unclassified. 92.5% of alleles have been characterized, and the mutational spectrum includes 36 different sequence variations presumably leading to loss-of-function, 15 changes in the BCKDHA, 14 in the BCKDHB, and seven in the DBT genes. Twenty-four changes are novel. The mutational profile is heterogeneous with no prevalent sequence variations detected, except for the E1beta mutation, c.487G>T (p.Glu163X), which appears on six out of 30 disease alleles analyzed. Approximately 30% of the patients included in this study showed a variant MSUD phenotype. That included 50% of the patients identified as EIa and at least four out of seven of those selected as EII. Precise genotypes as c.[647C>T]+[889C>T] for the EIa and c.[827T>G ]+[1349C>A] for the EII appeared associated to the mildest presentations of the disease.

Identification of two new sets of genes for dibenzothiophene transformation in Burkholderia sp. DBT1.

A novel genotype for the initial steps of the oxidative degradation of dibenzothiophene (DBT) is described in a Burkholderia sp. strain isolated from a drain receiving oil refinery wastewater. The strain is capable of transforming DBT with significant efficiency when compared to other microorganisms. Its genotype was discovered by investigating insertional mutants of genes involved in DBT degradation by the Kodama pathway. The cloned dbt genes show a novel genomic organization when compared to previously described genes capable of DBT catabolism in that they constitute two distinct operons and are not clustered in a single transcript. Sequence analysis suggests the presence of a sigma54-dependent positive transcriptional regulator that may be involved in the control of the transcription of the two operons, both activated by DBT. The achieved results suggest the possibility of novel features of DBT biotransformation in nature.

Maple syrup urine disease in the Austronesian aboriginal tribe Paiwan of Taiwan: a novel DBT (E2) gene 4.7 kb founder deletion caused by a nonhomologous recombination between LINE-1 and Alu and the carrier-frequency determination.

Maple syrup urine disease (MSUD) is an autosomal recessive inborn error disorder derived from the accumulation of the branched-chain amino acids (BCAAs) leucine, isoleucine and valine. Either the E1alpha, E1beta or DBT (E2) genes are responsible for this neurometabolic disease. Here, we report the identification and characterization of a novel E2 gene 4.7 kb deletion as a rare nonhomologous recombination of the long interspersed nuclear elements 1 (LINE-1) in intron 10 and the Alu in the 3' UTR of the E2 gene from three classic MSUD patients of the Austronesian aboriginal tribe Paiwan in Taiwan. The E2 gene 4.7 kb deletion accounted for five out of six alleles in the three unrelated Paiwanese MSUD patients, indicating a founder effect. Carrier-frequency study revealed one deleted heterozygote out of 101 normal Paiwanese. As the nine Taiwanese Austronesian aboriginal tribes share a common origin, this E2 4.7 kb deletion may be preserved in some of the other Austronesian aboriginal tribes of Taiwan. This is the first comprehensive genetics study of MSUD in the Austronesian tribal groups as well as in Taiwan.

Localization of the dihydrolipoamide branched-chain transacylase gene (DBT) of the human branched-chain keto acid dehydrogenase complex to chromosome 1

The gene coding for the transacylase subunit (DBT) of the human branched-chain keto acid dehydrogenase complex was localized to chromosome 1 by probing panels of human x mouse chromosome hybrids with an E2 cDNA amplified by the polymerase chain reaction. Additional data with two hybrids containing chromosome 1 fragments suggest that the DBT gene is located on the short arm (1pter----p21) of the chromosome.