Α-keto Acid Dehydrogenase Complex Research Articles

A Novel Regulatory Defect in the Branched-Chain α-Keto Acid Dehydrogenase Complex Due to a Mutation in thePPM1KGene Causes a Mild Variant Phenotype of Maple Syrup Urine Disease

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others.

Analysis of gene mutations in Chinese patients with maple syrup urine disease

Maple syrup urine disease (MSUD) is predominantly caused by mutations in the BCKDHA, BCKDHB and DBT genes, which encode for the E1α, E1β and E2 subunits of the branched-chain α-keto acid dehydrogenase complex, respectively. The aim of this study was to screen DNA samples from 16 Chinese MSUD patients and assess a potential correlation between genotype and phenotype. BCKDHA, BCKDHB and DBT genes were analyzed by polymerase chain reaction (PCR) and direct sequencing. Segments bearing novel mutations were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Within the variant alleles, 28 mutations (28/32, 87.5%), were detected in 15 patients, while one patient displayed no mutations. Mutations were comprised of 20 different: 6 BCKDHA gene mutations in 4 cases, 10 BCKDHB gene mutations in 8 cases and 4 DBT gene mutations in 3 cases. From these, 14 were novel, which included 3 mutations in the BCKDHA gene, 7 in the BCKDHB gene and 4 in the DBT gene. Only two patients with mutations in the BCKDHB and DBT genes were thiamine-responsive and presented a better clinical outcome. We identified 20 different mutations within the BCKDHA, BCKDHB and DBT genes among 16 Chinese MSUD patients, including 14 novel mutations. The majority were non-responsive to thiamine, associating with a worse clinical outcome. Our data provide the basis for further genotype-phenotype correlation studies in these patients, which will be beneficial for early diagnosis and in directing the approach to clinical intervention.

Characterization of a Naturally Occurring Mutation (Ile-358 to Thr) in Human Dihydrolipoamide Dehydrogenase of a Patient with Leigh Syndrome

E-mail: hjkim@daegu.ac.kr Received January 6, 2012, Accepted January 30, 2012Key Words : Dihydrolipoamide dehydrogenase, Pyridine nucleotide-disulfide oxidoreductase, Site-directedmutagenesis, Dihydrolipoamide deficiency, α-Keto acid dehydrogenase complexesDihydrolipoamide dehydrogenase (E3) is a common flavo-protein component in three α-keto acid dehydrogenasecomplexes, such as pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenase complexes.

Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells

Lactation is associated with elevated catabolism of branched-chain amino acids (BCAA) in mammary glands to produce glutamate, glutamine, alanine, aspartate, and asparagine. This study determined effects of metabolic fuels on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 0.5 mM L-leucine and either L-[1-(14)C]leucine or L-[U-(14)C]leucine. The medium also contained 0-5 mM D-glucose, 0-2 mM L-glutamine, 0-4 mM DL-β-hydroxybutyrate, or 0-2 mM oleic acid. Rates of leucine decarboxylation were 60 % lower, but rates of α-ketoisocaproate production were 34 % higher, in the presence of 2 mM glucose than in its absence. All variables of leucine catabolism did not differ between 2 and 5 mM glucose or between 0 and 4 mM DL-β-hydroxybutyrate. Compared with 0-0.25 mM glutamine, 0.5 and 2 mM L-glutamine reduced leucine transport, transamination, and decarboxylation. In contrast, increasing the concentration of oleic acid from 0 to 2 mM dose-dependently stimulated leucine transamination, decarboxylation, and oxidation of carbons 2-6. Oleic acid also enhanced the abundance of cytosolic BCAA transaminase, while reducing the phosphorylated level (inactive state) of the E1α subunit of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. Thus, hypoglycemia or ketosis in early lactation does not likely affect BCAA metabolism in mammary epithelial cells. Increasing circulating levels of BCAA and oleic acid may have great potential to increase the syntheses of glutamate, glutamine, aspartate, alanine, and asparagine by lactating mammary glands, thereby leading to enhanced production of milk for suckling neonates.

Antioxidant administration prevents memory impairment in an animal model of maple syrup urine disease

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder resulting from deficiency of branched-chain α-keto acid dehydrogenase complex leading to branched chain amino acids (BCAA) leucine, isoleucine, and valine accumulation as well as their corresponding transaminated branched-chain α-keto acids. MSUD patients present neurological dysfunction and cognitive impairment. Here, we investigated whether acute and chronic administration of a BCAA pool causes impairment of acquisition and retention of avoidance memory in young rats. We have used two administration protocols. Acute administration consisted of three subcutaneous administrations of the BCAA pool (15.8μL/g body weight at 1-h intervals) containing 190mmol/L leucine, 59mmol/L isoleucine, and 69mmol/L valine or saline solution (0.85% NaCl; control group) in 30 days old Wistar rats. Chronic administration consisted of two subcutaneous administrations of BCAA pool for 21 days in 7 days old Wistar rats. N-acetylcysteine (NAC; 20mg/kg) and deferoxamine (DFX; 20mg/kg) co administration influence on behavioral parameters after chronic BCAA administration was also investigated. BCAA administration induced long-term memory impairment in the inhibitory avoidance and CMIA (continuous multiple-trials step-down inhibitory avoidance) tasks whereas with no alterations in CMIA retention memory. Inhibitory avoidance alterations were prevented by NAC and DFX. BCAA administration did not impair the neuropsychiatric state, muscle tone and strength, and autonomous function evaluated with the SHIRPA (SmithKline/Harwell/ImperialCollege/RoyalHospital/Phenotype Assessment) protocol. Taken together, our results indicate that alterations of motor activity or emotionality probably did not contribute to memory impairment after BCAA administration and NAC and DFX effects suggest that cognition impairment after BCAA administration may be caused by oxidative brain damage.

Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn(2+) ions for phosphatase activity, whereas Mg(2+) and Ca(2+) ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nm and 10 μm for Mn(2+) and Mg(2+) ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca(2+)-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K(m) for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.

Two novel mutations in the BCKDHB gene (R170H, Q346R) cause the classic form of maple syrup urine disease (MSUD)

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. BCKD is a mitochondrial complex encoded by four nuclear genes (BCKDHA, BCKDHB, DBT, and DLD) and is involved in the metabolism of branched-chain amino acids (BCAAs). In this study, we investigated the DNA sequences of BCKDHA, BCKDHB and DBT genes for mutations in a Chinese newborn with the classic form of MSUD and predicted the associated conformational changes using molecular modeling. We identified two previously unreported mutations in the BCKDHB gene, R170H (c.509G>A) in exon 5 and Q346R (c.1037 A>G) in exon 9. In silico analysis of the two novel missense mutations revealed that the mutation R170H-β alters the spatial orientation with both Y195-β′ and S206-α, which results in unstable β–β′ assembly and an unstable K+ ion binding loop of the α subunit, respectively; The Q346R mutation is predicted to disrupt the spatial conformation between Q346-β and I357-β′, which reduces the affinity of the β–β′ subunits. These results indicate that R170-β and Q346-β are crucial for the activity of the E1 component. These two novel mutations, R170H and Q346R result in the patient's clinical manifestation of the classic form of MSUD.

Lipoic acid metabolism of Plasmodium--a suitable drug target.

α-Lipoic acid (6,8-thioctic acid; LA) is a vital co-factor of α-ketoacid dehydrogenase complexes and the glycine cleavage system. In recent years it was shown that biosynthesis and salvage of LA in Plasmodium are necessary for the parasites to complete their complex life cycle. LA salvage requires two lipoic acid protein ligases (LplA1 and LplA2). LplA1 is confined to the mitochondrion while LplA2 is located in both the mitochondrion and the apicoplast. LplA1 exclusively uses salvaged LA and lipoylates α-ketoglutarate dehydrogenase, branched chain α-ketoacid dehydrogenase and the H-protein of the glycine cleavage system. LplA2 cannot compensate for the loss of LplA1 function during blood stage development suggesting a specific function for LplA2 that has yet to be elucidated. LA salvage is essential for the intra-erythrocytic and liver stage development of Plasmodium and thus offers great potential for future drug or vaccine development.LA biosynthesis, comprising octanoyl-acyl carrier protein (ACP) : protein N-octanoyltransferase (LipB) and lipoate synthase (LipA), is exclusively found in the apicoplast of Plasmodium where it generates LA de novo from octanoyl-ACP, provided by the type II fatty acid biosynthesis (FAS II) pathway also present in the organelle. LA is the co-factor of the acetyltransferase subunit of the apicoplast located pyruvate dehydrogenase (PDH), which generates acetyl-CoA, feeding into FAS II. LA biosynthesis is not vital for intra-erythrocytic development of Plasmodium, but the deletion of several genes encoding components of FAS II or PDH was detrimental for liver stage development of the parasites indirectly suggesting that the same applies to LA biosynthesis.These data provide strong evidence that LA salvage and biosynthesis are vital for different stages of Plasmodium development and offer potential for drug and vaccine design against malaria.

Characterization of Three Site-specific Mutations in Human Dihydrolipoamide Dehydrogenase (Gly-42 to Ala, Gly-43 to Ala and Val-48 to Trp)

and catalyzes the reoxidation of the dihydrolipoyl prostheticgroup attached to the lysyl residue(s) of the acyltransferasecomponents of the three α-keto acid dehydrogenase com-plexes and to the hydrogen-carrier protein of the glycinecleavage system. Because E3 is a common component in thethree α-keto acid dehydrogenases, a decrease in E3 activitycan affect the activities of all three complexes, which canresult in increased urinary excretion of α-keto acids, elevatedblood lactate, pyruvate and branched chain amino acids. E3 belongs to the pyridine nucleotide-disulfide oxidore-ductase family along with glutathione reductase, thioredoxinreductase, mercuric reductase and trypanothione reductase.

Clofibrate‐Induced Reduction of Plasma Branched‐Chain Amino Acid Concentrations Impairs Glucose Tolerance in Rats

It has been reported that branched-chain amino acid (BCAA) administration stimulates glucose uptake into muscles and whole body glucose oxidation in rats. The authors examined the effect of decreased plasma BCAA concentrations induced by clofibrate treatment on glucose tolerance in rats. Since clofibrate, a drug for hyperlipidemia (high serum triglyceride concentration), is a potent inhibitor of the branched-chain α-keto acid dehydrogenase kinase, clofibrate treatment (0.2 g/kg body weight) activated the hepatic branched-chain α-keto acid dehydrogenase complex, resulting in decreased plasma BCAA concentrations by 30% to 50% from the normal level. An intraperitoneal glucose tolerance test was conducted after clofibrate administration, and the results showed that peak plasma glucose concentration and the area under the curve of glucose concentration during the intraperitoneal glucose tolerance test were significantly higher in clofibrate-treated rats than in control rats. This impaired glucose tolerance in the clofibrate-treated rats was ameliorated by administration of BCAAs (0.45 g/kg body weight, leucine:isoleucine:valine = 2:1:1), which kept plasma BCAA concentrations at normal levels during the intraperitoneal glucose tolerance test. These results suggest that plasma BCAAs play an important role in maintaining normal glucose tolerance in rats.

Reduced Expression of Lipoic Acid Synthase Accelerates Diabetic Nephropathy

Oxidative stress contributes to the pathogenesis of diabetic nephropathy. In mitochondria, lipoic acid synthase produces α-lipoic acid, an antioxidant and an essential cofactor in α-ketoacid dehydrogenase complexes, which participate in glucose oxidation and ATP generation. Administration of lipoic acid abrogates diabetic nephropathy in animal models, but whether lower production of endogenous lipoic acid promotes diabetic nephropathy is unknown. Here, we crossed mice heterozygous for lipoic acid synthase deficiency (Lias(+/-)) with Ins2(Akita/+) mice, a well characterized model of type 1 diabetes. Double mutant mice had more overt diabetic nephropathy, including microalbuminuria, glomerular basement thickening, mesangial matrix expansion, and hypertension, compared with Lias(+/+)Ins2(Akita/+) controls. We also identified proximal tubules as a major site for generation of superoxide anions during diabetic nephropathy. Mitochondria in proximal tubular cells were particularly sensitive to damage in diabetic mice with reduced lipoic acid production. These results suggest that lipoic acid synthase deficiency increases oxidative stress and accelerates the development of diabetic nephropathy.

Branched-chain amino acids (BCAAs) as nutraceuticals for exercise

Branched-chain amino acids (BCAAs: leucine, isoleucine and valine) are essential amino acids for humans and animals. It has been shown that BCAA oxidation is promoted by exercise through activation of branched-chain α-keto acid dehydrogenase complex (BCKDC), which is a rate-limiting enzyme in the catabolic pathway of the BCAAs, and the elevated enzyme activity in skeletal muscle is quickly downregulated after exercise. This tight regulation of the BCKDC is consistent with stimulation of protein synthesis after exercise in skeletal muscle. With this background, it is interesting to consider the BCAAs as a supplement for sports. We conducted a human study to examine the effects of BCAA supplementation on the delayed-onset muscle soreness (DOMS) induced by squatting exercises. The results clearly showed that BCAA supplementation significantly reduced DOMS evaluated by a visual analog scale method and maintained leg muscle force during the period of DOMS. Other studies using different types of exercise support our findings, suggesting that BCAA supplements are useful for conditioning of skeletal muscle after exercise.

Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain α-Ketoacid Dehydrogenase Complex

The purified mammalian branched-chain α-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain α-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-Å resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other α-ketoacid dehydrogenase complexes.

1,3-Dinitrobenzene–Induced Metabolic Impairment through Selective Inactivation of the Pyruvate Dehydrogenase Complex

Prolonged exposure to the chemical intermediate, 1,3-dinitrobenzene (1,3-DNB), produces neuropathology in the central nervous system of rodents analogous to that observed in various conditions of acute energy deprivation including thiamine deficiency and Leigh's necrotizing encephalopathy. Increased production of reactive intermediates in addition to induction of oxidative stress has been implicated in the neurotoxic mechanism of 1,3-DNB, but a clear metabolic target has not been determined. Here we propose that similar to thiamine deficiency, the effects of 1,3-DNB on metabolic status may be due to inhibition of the thiamine-dependent α-ketoacid dehydrogenase complexes. The effects of 1,3-DNB on astroglial metabolic status and α-ketoacid dehydrogenase activity were evaluated using rat C6 glioma cells. Exposure to 1,3-DNB resulted in altered morphology and biochemical dysfunction consistent with disruption of oxidative energy metabolism. Cotreatment with acetyl-carnitine or acetoacetate attenuated morphological and metabolic effects of 1,3-DNB exposure as well as increased cell viability. 1,3-DNB exposure inhibited pyruvate dehydrogenase complex (PDHc) and the inhibition correlated with the loss of lipoic acid (LA) immunoreactivity, suggesting that modification of LA is a potential mechanism of inhibition. Treatment with antioxidants and thiol-containing compounds failed to protect against loss of LA. Alternatively, inhibition of dihydrolipoamide dehydrogenase, the E3 component of the complex attenuated loss of LA. Collectively, these data suggest that 1,3-DNB impairs oxidative energy metabolism through direct inhibition of the PDHc and that this impairment is due to perturbations in the function of protein-bound LA.

Movement disorders in adult surviving patients with maple syrup urine disease

Maple syrup urine disease is a rare metabolic disorder caused by mutations in the branched-chain α-keto acid dehydrogenase complex gene. Patients generally present early in life with a toxic encephalopathy because of the accumulation of the branched-chain amino acids leucine, isoleucine, and valine and the corresponding ketoacids. Movement disorders in maple syrup urine disease have typically been described during decompensation episodes or at presentation in the context of a toxic encephalopathy, with complete resolution after appropriate dietary treatment. Movement disorders in patients surviving childhood are not well documented. We assessed 17 adult patients with maple syrup urine disease (mean age, 27.5 years) with a special focus on movement disorders. Twelve (70.6%) had a movement disorder on clinical examination, mainly tremor and dystonia or a combination of both. Parkinsonism and simple motor tics were also observed. Pyramidal signs were present in 11 patients (64.7%), and a spastic-dystonic gait was observed in 6 patients (35.2%). In summary, movement disorders are common in treated adult patients with maple syrup urine disease, and careful neurological examination is advisable to identify those who may benefit from specific therapy. © 2011 Movement Disorder Society.

Phenylbutyrate therapy for maple syrup urine disease

Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate in urea cycle disorder patients has been associated with a selective reduction in branched-chain amino acids (BCAA) in spite of adequate dietary protein intake. Based on this clinical observation, we investigated the potential of phenylbutyrate treatment to lower BCAA and their corresponding α-keto acids (BCKA) in patients with classic and variant late-onset forms of maple syrup urine disease (MSUD). We also performed in vitro and in vivo experiments to elucidate the mechanism for this effect. We found that BCAA and BCKA are both significantly reduced following phenylbutyrate therapy in control subjects and in patients with late-onset, intermediate MSUD. In vitro treatment with phenylbutyrate of control fibroblasts and lymphoblasts resulted in an increase in the residual enzyme activity, while treatment of MSUD cells resulted in the variable response which did not simply predict the biochemical response in the patients. In vivo phenylbutyrate increases the proportion of active hepatic enzyme and unphosphorylated form over the inactive phosphorylated form of the E1α subunit of the branched-chain α-keto acid dehydrogenase complex (BCKDC). Using recombinant enzymes, we show that phenylbutyrate prevents phosphorylation of E1α by inhibition of the BCKDC kinase to activate BCKDC overall activity, providing a molecular explanation for the effect of phenylbutyrate in a subset of MSUD patients. Phenylbutyrate treatment may be a valuable treatment for reducing the plasma levels of neurotoxic BCAA and their corresponding BCKA in a subset of MSUD patients and studies of its long-term efficacy are indicated.

Successful TAT-mediated enzyme replacement therapy in a mouse model of mitochondrial E3 deficiency

Medicine today offers no cure for patients suffering from mitochondrial disorders, such as lipoamide dehydrogenase (LAD; also known as E3) deficiency, and treatment is limited to symptomatic care. LAD is one of the components of the α-ketoacid dehydrogenase complexes, which are mitochondrial multienzyme complexes crucial for the metabolism of carbohydrates and amino acids. Recently, we tested the therapeutic approach for treating mitochondrial disorders whereby the activity of multicomponent complexes in the mitochondria is restored by TAT-mediated enzyme replacement therapy (ERT). The LAD deficiency disease was used before as a proof-of-principle in vitro, in patients' cells, utilizing the TAT-LAD fusion protein. In this report, we present successful TAT-mediated ERT in an in vivo mouse model using E3-deficient mice. We demonstrate the delivery of TAT-LAD into E3-deficient mice tissues and that a single administration of TAT-LAD results in a significant increase in the enzymatic activity of the mitochondrial multienzyme complex pyruvate dehydrogenase complex within the liver, heart and, most importantly, the brain of TAT-LAD-treated E3-deficient mice. We believe that this TAT-mediated ERT approach could change the management of mitochondrial disorders and of other metabolic diseases in modern medicine.

Ornish and Pritikin Programs Approved by CMS

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy caused by a deficiency in branched-chain α-keto acid dehydrogenase complex activity that leads to the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine, and valine and their respective α-keto-acids, α-ketoisocaproic acid (KIC), α keto-β-methylvaleric acid, and α-ketoisovaleric acid. The major clinical features presented by MSUD patients include ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay, and mental retardation; however, the pathophysiology of this disease is poorly understood. MSUD treatment consists of a low protein diet supplemented with a mixture containing micronutrients and essential amino acids but excluding BCAAs. Studies have shown that oxidative stress may be involved in the neuropathology of MSUD, with the existence of lipid and protein oxidative damage in affected patients. In recent years, studies have demonstrated the antioxidant role of l-carnitine (l-Car), which plays a central function in cellular energy metabolism and for which MSUD patients have a deficiency. In this work, we investigated the in vitro effect of Leu and KIC in the presence or absence of l-Car on DNA damage in peripheral whole blood leukocytes using the alkaline comet assay with silver staining and visual scoring. Leu and KIC resulted in a DNA damage index that was significantly higher than that of the control group, and l-Car was able to significantly prevent this damage, mainly that due to KIC.

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.

Functional characterization of the novel intronic nucleotide change c.288+9C>T within the BCKDHA gene: understanding a variant presentation of maple syrup urine disease

Mutations in any of the three different genes--BCKDHA, BCKDHB, and DBT--encoding for the E1α, E1β, and E2 catalytic components of the branched-chain α-ketoacid dehydrogenase complex can cause maple syrup urine disease (MSUD). Disease severity ranges from the classic to the mildest variant types and precise genotypes, mostly based on missense mutations, have been associated to the less severe presentations of the disease. Herein, we examine the consequences at the messenger RNA (mRNA) level of the novel intronic alteration c.288+9C>T found in heterozygous fashion in a BCKDHA variant MSUD patient who also carries the nucleotide change c.745G>A (p.Gly249Ser), previously described as a severe change. Direct analysis of the processed transcripts from the patient showed--in addition to a low but measurable level of normal mRNA product--an aberrantly spliced mRNA containing a 7-bp fragment of intron 2, which could be rescued when the patient's cells were treated with emetine. This aberrant transcript with a premature stop codon would be unstable, supporting the possible activation of nonsense-mediated mRNA decay pathway. Consistent with this finding, minigene splicing assays demonstrated that the point mutation c.288+9C>T is sufficient to create a cryptic splice site and cause the observed 7-bp insertion. Furthermore, our results strongly suggest that the c.288+9C>T allele in the patient generates both normal and aberrant transcripts that could sustain the variant presentation of the disease, highlighting the importance of correct genotyping to establish genotype-phenotype correlations and as basis for the development of therapeutic interventions.