An insight into the role of branched-chain α-keto acid dehydrogenase (BKD) complex in branched-chain fatty acid biosynthesis and virulence of Listeria monocytogenes.

Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of L. monocytogenes that becomes active upon the entry of the bacterium into the cytosol of infected cells. L. monocytogenes can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by lpd, bkdA1, bkdA2, and bkdB, is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient Listeria strains by in-frame deletion of lpd, bkdA1, bkdA2, and bkdB genes. To determine the role in in vivo and in vitro, mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in ΔbkdA1 strain than those in the wild-type. This study demonstrates that L. monocytogenes strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type prfA may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for L. monocytogenes to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between L. monocytogenes membrane integrity and the function of PrfA. This knowledge will increase our understanding of L. monocytogenes pathogenesis, which may provide insight into the development of antimicrobial agents.

Mitolnc controls cardiac BCAA metabolism and heart hypertrophy by allosteric activation of BCKDH.

Enzyme activity is determined by various different mechanisms, including posttranslational modifications and allosteric regulation. Allosteric activators are often metabolites but other molecules serve similar functions. So far, examples of long non-coding RNAs (lncRNAs) acting as allosteric activators of enzyme activity are missing. Here, we describe the function of mitolnc in cardiomyocytes, a nuclear encoded long non-coding RNA, located in mitochondria and directly interacting with the branched-chain ketoacid dehydrogenase (BCKDH) complex to increase its activity. The BCKDH complex is critical for branched-chain amino acid catabolism (BCAAs). Inactivation of mitolnc in mice reduces BCKDH complex activity, resulting in accumulation of BCAAs in the heart and cardiac hypertrophy via enhanced mTOR signaling. We found that mitolnc allosterically activates the BCKDH complex, independent of phosphorylation. Mitolnc-mediated regulation of the BCKDH complex constitutes an important additional layer to regulate the BCKDH complex in a tissue-specific manner, evading direct coupling of BCAA metabolism to ACLY-dependent lipogenesis.

Successful treatment of severe MSUD in Bckdhb-/- mice with neonatal AAV gene therapy.

Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb-/- mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha-/- mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb-/- mice at 1014 vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb-/- mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.

Structure-guided engineering of branched-chain α-keto acid decarboxylase for improved 1,2,4-butanetriol production by in vitro synthetic enzymatic biosystem.

Efficient synthetic routes for biomanufacturing chemicals often require the overcoming of pathway bottlenecks by tailoring enzymes to improve the catalytic efficiency or even implement non-native activities. 1,2,4-butanetriol (BTO), a valuable commodity chemical, is currently biosynthesized from D-xylose via a four-enzyme reaction cascade, with the ThDP-dependent α-keto acid decarboxylase (KdcA) identified as the potential bottleneck. Here, to further enhance the catalytic activity of KdcA toward the non-native substrate α-keto-3-deoxy-xylonate (KDX), in silico screening and structure-guided evolution were performed. The best mutants, S286L/G402P and V461K, exhibited a 1.8- and 2.5-fold higher enzymatic activity in the conversion of KDX to 3,4-dihydroxybutanal when compared to KdcA, respectively. MD simulations revealed that the two sets of mutations reshaped the substrate binding pocket, thereby increasing the binding affinity for KDX and promoting interactions between KDX and cofactor ThDP. Then, when the V461K mutant instead of wild type KdcA was integrated into the enzyme cascade, a 1.9-fold increase in BTO titer was observed. After optimization of the reaction conditions, the enzyme cocktail contained V461K converted 60 g/L D-xylose to 22.1 g/L BTO with a yield of 52.1 %. This work illustrated that protein engineering is a powerful tool for modifying the output of metabolic pathway.

Outcomes from a Single Transplant Center of 5 Pediatric Cases of Domino Liver Transplantation from Live Donors with Maple Syrup Urine Disease.

BACKGROUND Maple syrup urine disease (MSUD) is a rare genetic deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex that breaks down amino acids, resulting in multi-organ failure. This report is of 5 pediatric cases of domino liver transplantation (DLT) from live donors with MSUD from a single transplant center in Beijing. CASE REPORT All MSUD donors were confirmed to have disease-causing mutations in BCKDHA (branched-chain keto acid dehydrogenase E1, alpha polypeptide) or BCKDHB (branched-chain keto acid dehydrogenase E1, ß polypeptide) genes by peripheral blood whole-exon sequencing. Serum leucine and valine concentrations were significantly higher than normal values. Recipients ranged in age from 0.75 to 9 years old. Three patients underwent auxiliary liver transplantation, and the other children all underwent liver or partial liver transplantation. This case report was followed up for 25 to 79 months. The prognosis, growth, and development of patients were followed up. By the end of the last follow-up, all children had survived. All patients had normal serum leucine and valine concentrations after surgery. In case 1, portal vein stenosis post-operatively. In case 2, stenosis of hepatic artery and bile duct occurred. In case 5, hepatic artery and portal vein stenosis occurred, resulting in graft loss.   CONCLUSIONS The findings from our center support the findings from other pediatric liver transplant centers that liver transplantation using MSUD donors can have successful outcomes without the development of MSUD in the recipient.

Downregulation of extramitochondrial BCKDH and its uncoupling from AMP deaminase in type 2 diabetic OLETF rat hearts.

Systemic branched-chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long-Evans-Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched-chain α-keto acid dehydrogenase (BCKDH), a rate-limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long-Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH-E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3-E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3-BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3-BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.

PPM1K defects cause mild maple syrup urine disease: The second case in the literature.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by the insufficient catabolism of branched-chain amino acids. BCKDHA, BCKDHB, DBT, and DLD encode the subunits of the branched-chain α-ketoacid dehydrogenase complex, which is responsible for the catabolism of these amino acids. Biallelic pathogenic variants in BCKDHA, BCKDHB, or DBT are characteristic of MSUD. In addition, a patient with a PPM1K defect was previously reported. PPM1K dephosphorylates and activates the enzyme complex. We report a patient with MSUD with mild findings and elevated BCAA levels carrying a novel homozygous start-loss variant in PPM1K. Our study offers further evidence that PPM1K variants cause mild MSUD.

Genomic and biochemical analysis of repeatedly observed variants in DBT in individuals with maple syrup urine disease of Central American ancestry.

Maple syrup urine disease (MSUD) is an intoxication-type inherited metabolic disorder in which hyperleucinemia leads to brain swelling and death without treatment. MSUD is caused by branched-chain alpha-ketoacid dehydrogenase deficiency due to biallelic loss of the protein products from the genes BCKDHA, BCKDHB, or DBT, while a distinct but related condition is caused by loss of DLD. In this case series, eleven individuals with MSUD caused by two pathogenic variants in DBT are presented. All eleven individuals have a deletion of exon 2 (delEx2, NM_001918.3:c.48_171del); six individuals are homozygous and five individuals are compound heterozygous with a novel missense variant (NM_001918.5:c.916 T > C [p.Ser306Pro]) confirmed to be in trans. Western Blot indicates decreased amount of protein product in delEx2;c.916 T > C liver cells and absence of protein product in delEx2 homozygous hepatocytes. Ultrahigh performance liquid chromatography-tandem mass spectrometry demonstrates an accumulation of branched-chain amino acids and alpha-ketoacids in explanted hepatocytes. Individuals with these variants have a neonatal-onset, non-thiamine-responsive, classical form of MSUD. Strikingly, the entire cohort is derived from families who immigrated to the Washington, DC, metro area from Honduras or El Salvador suggesting the possibility of a founder effect.

Pathogenic Homozygous Mutations in the DBT Gene (c.1174A>C) Result in Maple Syrup Urine Disease in a rs12021720 Carrier.

OBJECTIVE: Maple syrup urine disease (MSUD; OMIM #248600) is an autosomal recessive metabolic disorder in the catabolism of branched-chain amino acids (leucine, isoleucine, and valine) and may be lethal if untreated in affected newborns. METHODS: Single-nucleotide polymorphism haplotyping and Sanger sequencing of BCKDHA, BCKDHB, and DBT genes were performed in a cohort of 10 MSUD patients. RESULTS: We identified a 16.6 Mb homozygous region harboring the DBT gene in an Iranian girl presenting with MSUD. Sanger sequencing revealed a pathogenic homozygous variant (NM_001918.3: c.1174A > C) in the DBT gene. We further found a controversial variant (rs12021720: c.1150 A > G) in the DBT gene. This substitution (p.Ser384Gly) is highly debated in literature. Bioinformatics and cosegregation analysis, along with identifying the real pathogenic variants (c.1174 A > C), lead to terminate these various interpretations of c.1150 A > G variant. CONCLUSION: Our study introduced c.1150 A > G as a polymorphic variant, which is informative for variant databases and also helpful in molecular diagnosis.

Neonatal gene therapy achieves sustained disease rescue of maple syrup urine disease in mice.

Maple syrup urine disease (MSUD) is a rare recessively inherited metabolic disorder causing accumulation of branched chain amino acids leading to neonatal death, if untreated. Treatment for MSUD represents an unmet need because the current treatment with life-long low-protein diet is challenging to maintain, and despite treatment the risk of acute decompensations and neuropsychiatric symptoms remains. Here, based on significant liver contribution to the catabolism of the branched chain amino acid leucine, we develop a liver-directed adeno-associated virus (AAV8) gene therapy for MSUD. We establish and characterize the Bckdha (branched chain keto acid dehydrogenase a)-/- mouse that exhibits a lethal neonatal phenotype mimicking human MSUD. Animals were treated at P0 with intravenous human BCKDHA AAV8 vectors under the control of either a ubiquitous or a liver-specific promoter. BCKDHA gene transfer rescued the lethal phenotype. While the use of a ubiquitous promoter fully and sustainably rescued the disease (long-term survival, normal phenotype and correction of biochemical abnormalities), liver-specific expression of BCKDHA led to partial, though sustained rescue. Here we show efficacy of gene therapy for MSUD demonstrating its potential for clinical translation.

A Gain-of-Function Mutation on BCKDK Gene and Its Possible Pathogenic Role in Branched-Chain Amino Acid Metabolism.

BCKDK is an important key regulator of branched-chain ketoacid dehydrogenase complex activity by phosphorylating and so inactivating branched-chain ketoacid dehydrogenases, the rate-limiting enzyme of the branched-chain amino acid metabolism. We identified, by whole exome-sequencing analysis, the p.His162Gln variant of the BCKDK gene in a neonate, picked up by newborn screening, with a biochemical phenotype of a mild form of maple syrup urine disease (MSUD). The same biochemical and genetic picture was present in the father. Computational analysis of the mutation was performed to better understand its role. Extensive atomistic molecular dynamics simulations showed that the described mutation leads to a conformational change of the BCKDK protein, which reduces the effect of inhibitory binding bound to the protein itself, resulting in its increased activity with subsequent inactivation of BCKDC and increased plasmatic branched-chain amino acid levels. Our study describes the first evidence of the involvement of the BCKDK gene in a mild form of MSUD. Although further data are needed to elucidate the clinical relevance of the phenotype caused by this variant, awareness of this regulatory activation of BCKDK is very important, especially in newborn screening data interpretation.

Novel Loss of Function Variant in BCKDK Causes a Treatable Developmental and Epileptic Encephalopathy.

Branched-chain amino acids (BCAA) are essential amino acids playing crucial roles in protein synthesis and brain neurotransmission. Branched-chain ketoacid dehydrogenase (BCKDH), the flux-generating step of BCAA catabolism, is tightly regulated by reversible phosphorylation of its E1α-subunit. BCKDK is the kinase responsible for the phosphorylation-mediated inactivation of BCKDH. In three siblings with severe developmental delays, microcephaly, autism spectrum disorder and epileptic encephalopathy, we identified a new homozygous in-frame deletion (c.999_1001delCAC; p.Thr334del) of BCKDK. Plasma and cerebrospinal fluid concentrations of BCAA were markedly reduced. Hyperactivity of BCKDH and over-consumption of BCAA were demonstrated by functional tests in cells transfected with the mutant BCKDK. Treatment with pharmacological doses of BCAA allowed the restoring of BCAA concentrations and greatly improved seizure control. Behavioral and developmental skills of the patients improved to a lesser extent. Importantly, a retrospective review of the newborn screening results allowed the identification of a strong decrease in BCAA concentrations on dried blood spots, suggesting that BCKDK is a new treatable metabolic disorder probably amenable to newborn screening programs.

Association of BCAT2 and BCKDH polymorphisms with clinical, anthropometric and biochemical parameters in young adults.

BACKGROUND AND AIM: Circulating amino acids are modified by sex, body mass index (BMI) and insulin resistance (IR). However, whether the presence of genetic variants in branched-chain amino acid (BCAA) catabolic enzymes modifies circulating amino acids is still unknown. Thus, we determined the frequency of two genetic variants, one in the branched-chain aminotransferase 2 (BCAT2) gene (rs11548193), and one in the branched-chain ketoacid dehydrogenase (BCKDH) gene (rs45500792), and elucidated their impact on circulating amino acid levels together with clinical, anthropometric and biochemical parameters. METHODS AND RESULTS: We performed a cross-sectional comparative study in which we recruited 1612 young adults (749 women and 863 men) aged 19.7 ± 2.1 years and with a BMI of 24.9 ± 4.7 kg/m2. Participants underwent clinical evaluation and provided blood samples for DNA extraction and biochemical analysis. The single nucleotide polymorphisms (SNPs) were determined by allelic discrimination using real-time polymerase chain reaction (PCR). The frequencies of the less common alleles were 15.2 % for BCAT2 and 9.83 % for BCKDH. The subjects with either the BCAT2 or BCKDH SNPs displayed no differences in the evaluated parameters compared with subjects homozygotes for the most common allele at each SNP. However, subjects with both SNPs had higher body weight, BMI, blood pressure, glucose, and circulating levels of aspartate, isoleucine, methionine, and proline than the subjects homozygotes for the most common allele (P < 0.05, One-way ANOVA). CONCLUSION: Our findings suggest that the joint presence of both the BCAT2 rs11548193 and BCKDH rs45500792 SNPs induces metabolic alterations that are not observed in subjects without either SNP.

Genetic analysis by targeted next-generation sequencing and novel variation identification of maple syrup urine disease in Chinese Han population.

Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder that affects the degradation of branched chain amino acids (BCAAs). Only a few cases of MSUD have been documented in Mainland China. In this report, 8 patients (4 females and 4 males) with MSUD from 8 unrelated Chinese Han families were diagnosed at the age of 6 days to 4 months. All the coding regions and exon/intron boundaries of BCKDHA, BCDKHB, DBT and DLD genes were analyzed by targeted NGS in the 8 MSUD pedigrees. Targeted NGS revealed 2 pedigrees with MSUD Ia, 5 pedigrees with Ib, 1 pedigree with MSUD II. Totally, 13 variants were detected, including 2 variants (p.Ala216Val and p.Gly281Arg) in BCKDHA gene, 10 variants (p.Gly95Ala, p.Ser171Pro, p.Phe175Leu, p.Arg183Trp, p.Lys222Thr, p.Arg285Ter, p.Arg111Ter, p.S184Pfs*46, p.Arg170Cys, p.I160Ffs*25) in BCKDHB gene, 1 variant (p.Arg431Ter) in DBT gene. In addition, 4 previously unidentified variants (p.Gly281Arg in BCKDHA gene, p.Ser171Pro, p.Gly95Ala and p.Lys222Thr in BCKDHB gene) were identified. NGS plus Sanger sequencing detection is effective and accurate for gene diagnosis. Computational structural modeling indicated that these novel variations probably affect structural stability and considered as likely pathogenic variants.

Maple syrup urine disease: Characteristics of diagnosis and treatment in 45 patients in Chile.

Maple urine syrup disease (MSUD) is an autosomal recessive disorder characterized by deficient activity of the branched-chain alpha ketoacid dehydrogenase (BCKAD) enzymatic complex due to biallelic variants in the alpha (BCKDHA) or beta (BCKDHB) subunits or the acyltransferase component (DBT). Treatment consists in leucine (LEU), isoleucine (ILE), and valine (VAL) (branched-chain amino acids) dietary restriction and strict metabolic control. to determine the characteristics of the Chilean cohort with MSUD currently in follow-up at Instituto de Nutrición y Tecnología de los Alimentos, during the 1990-2017 period Retrospective analytical study in 45 MSUD cases. Measured: biochemical parameters (LEU, ILE, and VAL), anthropometric evaluation, and neurocognitive development. In 18 cases undergoing genetic study were analyzed according to the gene and protein location, number of affected alleles, and type of posttranslational modification affected. Then, 45 patients with MSUD diagnosis were identified during the period: 37 were alive at the time of the study. Average diagnosis age was 71 ± 231 days. Average serum diagnosis LEU concentrations: 1.463 ± 854.1 µmol/L, VAL 550 ± 598 µmol/L and ILE 454 ± 458 µmol/L. BCKDHB variants explain 89% cases, while BCKDHA and DBT variants explain 5.5% of cases each. Variants p.Thr338Ile in BCKDHA, p.Pro240Thr and p.Ser342Asn in BCKDHB have not been previously reported in literature. Average serum follow-up LEU concentrations were 252.7 ± 16.9 µmol/L in the <5 years group and 299 ± 123.2 µmol/L in ≥5 years. Most cases presented some degree of developmental delay. Early diagnosis and treatment is essential to improve the long-term prognosis. Frequent blood LEU measurements are required to optimize metabolic control and to establish relationships between different aspects analyzed.

Superior production of heavy pamamycin derivatives using a bkdR deletion mutant of Streptomyces albus J1074/R2.

BACKGROUND: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. RESULTS: Supplemented with L-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post L-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. CONCLUSIONS: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

Neonatal maple syrup urine disease in China: two novel mutations in the BCKDHB gene and literature review.

OBJECTIVES: To report two novel mutations in the BCKDHB gene with Maple syrup urine disease (MSUD) and compare their data with 52 cases of MSUD reported in the available Chinese literature. METHODS: Clinical data of a case of a newborn with MSUD was retrospectively studied. Literatures on MSUD in the local medical journals from January 1990 till December 2019 in China were reviewed. RESULTS: Two novel BCKDHB mutations c.90_91insCTGGCGCGGGG (p.Phe35TrpfsTer41) and c.80_90del (p.Ala32PhefsTer48) were identified. We found a total of 52 cases of MSUD reports so far. A total of 49 cases had the symptom of poor feeding (94.2%), 50 cases showed poor responses to stimulation (96.2%), 21 cases had odor of maple syrup (40.3%), 29 cases had seizures (55.7%), and 13 cases had respiratory failure (25.0%). The average of the blood ammonia was 127.2 ± 75.0 µmol/L. A total of 18 cases reported the gene testing, among of them 9 cases of BCKDHA mutations, 6 cases of BCKDHB mutations, and 2 cases of DBT mutations. A total of 13 cases (25%) were treated with mechanical ventilation, 50 cases (96.2%) with protein-restricted diet and l-carnitine, 29 cases with thiamine, and only 2 cases were treated with blood purification. Finally, 19 patients (36.5%) were died, 21 cases (40.4%) were improved after treatments. CONCLUSIONS: The clinical phenotype of neonatal MSUD in China belongs to the classical type currently. Suspected patients should have blood or urine branched-chain amino acid levels tested and brain MRI as early as possible to enable early diagnosis, thus improvement in prognosis.

Molecular basis of various forms of maple syrup urine disease in Chilean patients.

BACKGROUND: Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disorder caused by the deficient activity of the branched-chain α-keto acid dehydrogenase (BCKD) enzymatic complex. BCKD is a mitochondrial complex encoded by four genes: BCKDHA, BCKDHB, DBT, and DLD. MSUD is predominantly caused by mutations in the BCKDHA, BCKDHB, and DBT genes which encode the E1α, E1ß, and E2 subunits of the BCKD complex, respectively. The aim of this study was to characterize the genetic basis of MSUD in a cohort of Chilean MSUD patients by identifying point mutations in the BCKDHA, BCKDHB, and DBT genes and to describe their impact on the phenotypic heterogeneity of these patients. METHODS: This manuscript describes a cross-sectional study of 18 MSUD patients carried out using PCR and DNA sequencing. RESULTS: Four novel pathogenic mutations were identified: one in BCKDHA (p.Thr338Ile), two in BCKDHB (p.Gly336Ser e p.Pro240Thr), and one in DBT (p.Gly406Asp). Four additional pathogenic mutations found in this study have been described previously. There were no correlations between the genotype and phenotype of the patients. CONCLUSION: If MSUD is diagnosed earlier, with a newborn screening approach, it might be possible to establish genotype-phenotype relationships more efficiently.

Serum amino acid concentrations are modified by age, insulin resistance, and BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms in subjects with obesity.

BACKGROUND & AIMS: The amino acid profile of young adults is modified by sex, body mass index (BMI) and insulin resistance (IR). However, we do not know if age or the presence of specific polymorphisms in the genes of BCAT2 and BCKDH contribute to changes in the amino acid profile, especially in subjects with obesity. Therefore, we have evaluated the effect of age, the presence of IR and the polymorphisms of BCAT2 rs11548193 and BCKDH rs45500792 on the concentration of amino acids in subjects with obesity. METHODS: This was a cross-sectional study conducted with 487 subjects with obesity. Participants underwent a physical examination in which their clinical history was obtained and a blood sample was taken for biochemical, hormonal, and DNA analysis. RESULTS: Adults <30 years old with obesity had higher levels of alanine, arginine, aspartate, histidine, leucine, lysine, methionine, phenylalanine, proline, serine and valine than adults ≥30 years old. Interestingly, regardless of age, we found that arginine, aspartate, serine decreased, while proline and tyrosine increased in the presence of IR; tyrosine and sum of branched-chain amino acids (∑BCAA) were the amino acids that increased in the presence of BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms. CONCLUSIONS: We found that the amino acid profiles of subjects with obesity are differentially modified by age, the presence of IR, and the presence of the BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms.

Effects of ketoisocaproic acid and inflammation on glucose transport in muscle cells.

Branched-chain amino acids (BCAAs) are regulators of protein metabolism. However, elevated levels of BCAAs and their metabolites are linked to insulin resistance. We previously demonstrated that the leucine metabolite, α-ketoisocaproate (KIC), inhibited insulin-stimulated glucose transport in myotubes. Like KIC, inflammatory factors are implicated in the development of insulin resistance. Here, we analyzed the effect of KIC and inflammatory factors (homocysteine [50 µM], TNF-α [10 ng/ml], and interleukin 6 (IL-6) [10 ng/ml]) on myotubes. Although KIC suppressed insulin-stimulated glucose transport, addition of the inflammatory factors did not worsen this effect. Depletion of branched-chain aminotransferase 2, the enzyme that catalyzes the conversion of leucine into KIC, abrogated the effect of KIC and the inflammatory factors. The effect of insulin on AKTS473 and S6K1T389 phosphorylation was not modified by treatments. There were no treatment effects on glycogen synthase phosphorylation. Depletion of E1α subunit of branched-chain α-keto acid dehydrogenase, the enzyme that catalyzes the oxidative decarboxylation of KIC, suppressed insulin-stimulated glucose transport, especially in cells incubated in KIC. Thus, defects in BCAA catabolism are contributory to insulin resistance of glucose transport in myotubes, especially in the presence of KIC. Interventions that increase BCAA catabolism may promote muscle glucose utilization and improve insulin resistance and its sequelae.