Molecular basis of maple syrup urine disease: novel mutations at the E1 alpha locus that impair E1(alpha 2 beta 2) assembly or decrease steady-state E1 alpha mRNA levels of branched-chain alpha-keto acid dehydrogenase complex.

We report the occurrence of three novel mutations in the E1 alpha (BCKDHA) locus of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1 alpha gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1 alpha subunit. Both the 8-bp deletion and the single C insertion generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1 alpha mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1 alpha subunit impairs its proper assembly with the normal E1 beta. Unassembled as well as misassembled E1 alpha and E1 beta subunits are degraded in the cell.     

Occurrence of a 2-bp (AT) deletion allele and a nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine disease: multiple-exon skipping as a secondary effect of the mutations.

We have identified two novel mutant alleles in the transacylase (E2) gene of the human branched-chain alpha-keto acid dehydrogenase (BCKAD) complex in 6 of 38 patients with maple syrup urine disease (MSUD). One mutation, a 2-bp (AT) deletion in exon 2 of the E2 gene, causes a frameshift downstream of residue (-26) in the mitochondrial targeting presequence. The second mutation, a G-to-T transversion in exon 6 of the E2 gene, produces a premature stop codon at Glu-163 (E163*). Transfection of constructs harboring the E163* mutation into an E2-deficient MSUD cell line produced a truncated E2 subunit. However, this mutant E2 chain is unable to assemble into a 24-mer cubic structure and is degraded in the cell. The 2-bp (AT) deletion and the E163* mutant alleles occur in either the homozygous or compound-heterozygous state in the 6 of 38 unrelated MSUD patients studied. Moreover, an array of precise single- and multiple-exon deletions were observed in many amplified E2 mutant cDNAs. The latter results appear to represent secondary effects on RNA processing that are associated with the MSUD mutations at the E2 locus.     

Structural and biochemical basis for novel mutations in homozygous Israeli maple syrup urine disease patients: a proposed mechanism for the thiamin-responsive phenotype

Maple syrup urine disease (MSUD) results from mutations affecting different subunits of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. In this study, we identified seven novel mutations in MSUD patients from Israel. These include C219W-alpha (TGC to TGG) in the E1alpha subunit; H156Y-beta (CAT to TAT), V69G-beta (GTT to GGT), IVS 9 del[-7:-4], and 1109 ins 8bp (exon 10) in the E1beta subunit; and H391R (CAC to CGC) and S133stop (TCA to TGA) affecting the E2 subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Recombinant E1 proteins carrying the C219W-alpha or H156Y-beta mutation show no catalytic activity with defective subunit assembly and reduced binding affinity for cofactor thiamin diphosphate. The mutant E1 harboring the V69G-beta substitution cannot be expressed, suggesting aberrant folding caused by this mutation. These E1 mutations are ubiquitously associated with the classic phenotype in homozygous-affected patients. The H391R substitution in the E2 subunit abolishes the key catalytic residue that functions as a general base in the acyltransfer reaction, resulting in a completely inactive E2 component. However, wild-type E1 activity is enhanced by E1 binding to this full-length mutant E2 in vitro. We propose that the augmented E1 activity is responsible for robust thiamin responsiveness in homozygous patients carrying the H391R E2 mutation and that the presence of a full-length mutant E2 is diagnostic of this MSUD phenotype. The present results offer a structural and biochemical basis for these novel mutations and will facilitate DNA-based diagnosis for MSUD in the Israeli population.     

A T-to-A substitution in the E1 alpha subunit gene of the branched-chain alpha-ketoacid dehydrogenase complex in two cell lines derived from Menonite maple syrup urine disease patients

We cloned and sequenced cDNAs of the E1 alpha and E1 beta subunits of the branched chain alpha-ketoacid dehydrogenase complex (BCKDH) in two cell lines derived from two different Menonite MSUD patients (GM 1655, GM 1099). A T-to-A substitution which generates an asparagine in place of a tyrosine at amino acid 394 of the mature E1 alpha subunit was present in both alleles in these two cell lines, whereas cDNAs of the E1 beta subunit in these cell lines were identical to that of normal human lymphoid cell line and that of the clone from a human placenta cDNA library. It is suggested that the Menonite MSUD is caused by the missense mutation of the E1 alpha subunit of the BCKDH complex.     

Complementation analysis in lymphoid cells from five patients with different forms of maple syrup urine disease

Summary The possibility of genetic heterogeneity in maple syrup urine discase was investigated by measuring branchedchain ketoacid dehydrogenase in polyethylene glycol-induced heterokaryons of lymphoid cells. The lymphoid cell lines from five patients with varying forms of the disease were establisjed after incubation with Epstein-Barr virus. The results suggested that there are at least two genetic complementation groups in the disease.This study was supported by a scientific research grant from the Ministry of Education, Science, and Culture of Japan (56480191; Matsuda) and a research grant from the Ministry of Health and Welfare of Japan     

Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD)

Skvorak et al. [1] demonstrated the therapeutic efficacy of HTx in a murine model of iMSUD, confirming significant metabolic improvement and survival. To determine the effect of HTx on extrahepatic organs, we examined the metabolic effects of HTx in brain from iMSUD animals. Amino acid analysis revealed that HTx corrected increased ornithine, partially corrected depleted glutamine, and revealed a trend toward alloisoleucine correction. For amino acid and monoamine neurotransmitters, decreased GABA was partially corrected with HTx, while the l-histidine dipeptide of GABA, homocarnosine, was decreased in iMSUD mice and hypercorrected following HTx. Elevated branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in MSUD can deplete brain tyrosine and tryptophan (the precursors of monoamine neurotransmitters, dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT)) through competition via the large neutral amino acid transporter. HTx corrected decreased DA levels and the DA metabolite, 3-methoxytyramine, and partially corrected the DA intermediate 3,4-dihydroxyphenylacetate (DOPAC) and 5-HT levels, despite normal tyrosine and tryptophan levels in iMSUD mouse brain. We further observed enhanced intracellular turnover of both DA and 5-HT in iMSUD mouse brain, both of which partially corrected with HTx. Our results suggest new pathomechanisms of neurotransmitter metabolism in this disorder and support the therapeutic relevance of HTx in iMSUD mice, while providing proof-of-principle that HTx has corrective potential in extrahepatic organs.     

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.     

A 17-bp insertion and a Phe215----Cys missense mutation in the dihydrolipoyl transacylase (E2) mRNA from a thiamine-responsive maple syrup urine disease patient WG-34.

We have amplified the cDNA for the transacylase (E2) subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex from a thiamine-responsive MSUD cell line (WG-34) by the polymerase chain reaction. Sequencing of the amplified WG-34 cDNA showed a 17-bp insertion (AAATACCTTGTTACCAG) apparently resulting from an aberrant splicing of the E2 gene, and a missense (T----G) mutation that changes Phe215 to Cys in the E2 subunit. The existence of these two mutations was confirmed by probing the amplified E2 cDNA or genomic DNA with allele-specific oligonucleotides. The above results support the thesis that the thiamine-responsive MSUD patient (WG-34) is a compound heterozygote at the E2 locus. The implication of the E2 mutations for the thiamine-responsiveness observed in this patient is discussed.     

Crystal structure of human branched-chain alpha-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease

BACKGROUND: Mutations in components of the extraordinarily large alpha-ketoacid dehydrogenase multienzyme complexes can lead to serious and often fatal disorders in humans, including maple syrup urine disease (MSUD). In order to obtain insight into the effect of mutations observed in MSUD patients, we determined the crystal structure of branched-chain alpha-ketoacid dehydrogenase (E1), the 170 kDa alpha(2)beta(2) heterotetrameric E1b component of the branched-chain alpha-ketoacid dehydrogenase multienzyme complex. RESULTS: The 2.7 A resolution crystal structure of human E1b revealed essentially the full alpha and beta polypeptide chains of the tightly packed heterotetramer. The position of two important potassium (K(+)) ions was determined. One of these ions assists a loop that is close to the cofactor to adopt the proper conformation. The second is located in the beta subunit near the interface with the small C-terminal domain of the alpha subunit. The known MSUD mutations affect the functioning of E1b by interfering with the cofactor and K(+) sites, the packing of hydrophobic cores, and the precise arrangement of residues at or near several subunit interfaces. The Tyr-->Asn mutation at position 393-alpha occurs very frequently in the US population of Mennonites and is located in a unique extension of the human E1b alpha subunit, contacting the beta' subunit. CONCLUSIONS: Essentially all MSUD mutations in human E1b can be explained on the basis of the structure, with the severity of the mutations for the stability and function of the protein correlating well with the severity of the disease for the patients. The suggestion is made that small molecules with high affinity for human E1b might alleviate effects of some of the milder forms of MSUD.     

Occurrence of a Tyr393----Asn (Y393N) mutation in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex in maple syrup urine disease patients from a Mennonite population

Maple syrup urine disease (MSUD) is caused by a deficiency in the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The incidence of MSUD in the Philadelphia Mennonites is 1/176 births resulting from consanguinity. In this study, we amplified cDNAs for the decarboxylase E1 alpha subunit of the branched-chain alpha-keto acid dehydrogenase complex from a classical MSUD patient and from an obligatory heterozygote of a Mennonite family by the PCR. Sequencing of the amplified cDNAs disclosed at codon 393 of the mature E1 alpha polypeptide a base substitution changing a tyrosine (encoded by TAC) to an asparagine residue (encoded by AAC), which is designated Y393N. A segment of the E1 alpha gene containing the 5' portion of exon 9 was amplified. Probing of the amplified genomic DNA with allele-specific oligonucleotide probes showed that the mutation in the E1 alpha gene was homozygous in six Mennonites affected with classical MSUD and was present in heterozygous carriers. The identification of the MSUD mutation in the Philadelphia Mennonites will facilitate diagnosis and carrier detection for this population.     

On the mechanisms of the formation of L-alloisoleucine and the 2-hydroxy-3-methylvaleric acid stereoisomers from L-isoleucine in maple syrup urine disease patients and in normal humans.

2-Keto-3-methylvaleric acid (KMVA) has been found not to undergo spontaneous keto-enol tautomerization in neutral aqueous solution, alone or in the presence of large concentrations of pyridoxamine or pyridoxamine-5-phosphate. This finding denies the commonly held suppositions that 3R-KMVA is derived spontaneously from 3S-KMVA in vivo, and that L-alloisoleucine is the product of the reamination of this 3R-KMVA. Evidence presented here suggests that racemization of the 3-carbon of L-isoleucine occurs during transamination, that L-alloisoleucine is an inherently unavoidable by-product of L-isoleucine transamination (and vice versa), and that a KMVA enol is not obligate in this racemization. The four stereoisomers of 2-hydroxy-3-methylvaleric acid have been synthesized and the mass spectra of their trimethylsilyl derivatives recorded. An achiral methylsilicone column was used to separate the diastereomeric pairs and to determine their relative ratios in plasma and urine from normal controls and two maple syrup urine disease (MSUD) patients. The urinary ratio of the two diastereomers is different from that for plasma, both in normals and in MSUD patients. The plasma ratios may provide a rapid and simple measure of residual branched chain 2-keto acid dehydrogenase activity in MSUD patients.     

Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.     

Reversion of the maple syrup urine disease phenotype of impaired branched chain alpha-ketoacid dehydrogenase complex activity in fibroblasts from an affected child

Branched chain alpha-ketoacid dehydrogenase is a heteroprotein complex of mitochondria and commits the branched chain alpha-ketoacids to their catabolic fate. Inherited nuclear mutations in humans decrease the activity of this complex and result in maple syrup urine disease. Here we demonstrate the restoration of branched chain alpha-ketoacid dehydrogenase activity to fibroblasts from a child with this disorder by transfection with a cDNA for the prebranched chain acyltransferase. Prior to transfection these fibroblasts contained the prebranched chain acyltransferase gene but failed to transcribe the gene and thus lacked the protein. Regulation of the restored complex by phosphorylation mechanisms resembles that of wild-type cells. These results describe a human cell modeling system for testing engineered proteins and support the possibility of gene replacement therapy for this human disorder.     

A new mutation of 5-alpha-reductase type 2 (A62E) in a large Egyptian kindred

OBJECTIVE: To describe the clinical, biological and molecular data in a large Egyptian kindred with 5alpha-reductase deficiency. PATIENTS AND METHODS: Three patients with ambiguous genitalia were referred at the ages of 20, 9 and 2 years, respectively. In all cases, parents were first cousins. Basal and post-HCG stimulation plasma levels of testosterone and dihydrotestosterone were determined. Direct sequencing and restriction site analysis were applied for patient and family study. RESULTS: A homozygous alanine to glutamic acid substitution at position 62 (A62E) was found in the three patients. The parents and two XX sisters were heterozygous while a third XX sibling was normal. CONCLUSION: We report a new mutation of the 5alpha-reductase type 2 gene. The presence of this mutation in all studied patients and their parents suggests its causative role in 5alpha-reductase deficiency. Identification of the mutation enabled genetic counselling for three XX individuals.     

Total branched-chain amino acids requirement in patients with maple syrup urine disease by use of indicator amino acid oxidation with L-[1-13C]phenylalanine

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defects in the mitochondrial multienzyme complex branched-chain alpha-keto acid dehydrogenase (BCKD; EC 1.2.4.4), responsible for the oxidative decarboxylation of the branched-chain ketoacids (BCKA) derived from the branched-chain amino acids (BCAA) leucine, valine, and isoleucine. Deficiency of the enzyme results in increased concentrations of the BCAA and BCKA in body cells and fluids. The treatment of the disease is aimed at keeping the concentration of BCAA below the toxic concentrations, primarily by dietary restriction of BCAA intake. The objective of this study was to determine the total BCAA requirements of patients with classical MSUD caused by marked deficiency of BCKD by use of the indicator amino acid oxidation (IAAO) technique. Five MSUD patients from the MSUD clinic of The Hospital for Sick Children participated in the study. Each was randomly assigned to different intakes of BCAA mixture (0, 20, 30, 50, 60, 70, 90, 110, and 130 mg.kg(-1).day(-1)), in which the relative proportion of BCAA was the same as that in egg protein. Total BCAA requirement was determined by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO(2). The mean total BCAA requirement was estimated using a two-phase linear regression crossover analysis, which showed that the mean total BCAA requirement was 45 mg.kg(-1).day(-1), with the safe level of intake (upper 95% confidence interval) at 62 mg.kg(-1).day(-1). This is the first time BCAA requirements in patients with MSUD have been determined directly.