Yoshitaka Nobukuni

Mutations in the PIT-1 gene in children with combined pituitary hormone deficiency

Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone and prolactin genes. In three unrelated Japanese children with combined pituitary hormone deficiency, we identified three point mutations in the Pit-1 gene, Pro24Leu, Arg143Gln, and Arg271Trp, located on the major transactivation region, POU-specific domain, and POU-homeodomain, respectively.

Characterization of the gene encoding human pituitary-specific transcription factor, Pit-1

Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone- and prolactin-encoding genes. A chromosomal gene related to human Pit-1 isolated from human gene libraries was over 14 kb long and split into six exons. All of the splice donor and acceptor sites conformed to the GT/AG rule. The gene was mapped to human chromosome region 3p11.

A T-to-A substitution in the E1α subunit gene of the branched-chain α-ketoacid dehydrogenase complex in two cell lines derived from menonite maple syrup urine disease patients

Abstract We cloned and sequenced cDNAs of the E 1 α and E 1 β subunits of the branched chain α-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 E 1 α subunit was present in both alleles in these two cell lines, whereas cDNAs of the E 1 β 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 E 1 α subunit of the BCKDH complex.

Complete primary structure of the transacylase (E2b) subunit of the human branched chain α-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.

Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp.

Gene analysis of mennonite maple syrup urine disease kindred using primer-specified restriction map modification

SummaryMaple syrup urine disease (MSUD) is an autosomal recessive inherited disease due to a deficiency of any of the subunits, E1α, E1β or E2, of the branched-chain α-ketoacid dehydrogenase complex (BCKDH). A large Mennonite kindred of MSUD has been studied in Pennsylvania, USA. In the present investigation, genomes from 70 members, including 12 patients belonging to eight different Mennonite MSUD pedigrees, were examined for possible abnormalities in the E1α gene of BCKDH, by primer-specified restriction map modification. A T-to-A substitution which generates an asparagine in place of a tyrosine at amino acid 394 of the mature E1α subunit was present in both alleles in all the patients and in a single allele in all obligate carriers and several siblings. We describe a new technique for rapid and easy detection of the mutant gene in this population. These family studies provide additional evidence that Mennonite MSUD is caused by a missense mutation of the E1α gene of BCKDH.

Maple syrup urine disease: a possible biochemical basis for the clinical heterogeneity

SummaryNine patients with maple syrup urine disease (MSUD), of whom eight were detected by mass-screening of neonates for inherited metabolic desease, were studied to determine possible relationships between clinical features and properties of the branched-chain α-keto acid dehydrogenase complex (BCKDH) in cultured lymphoblastoid cells. Based on their tolerance for leucine and on the clinical manifestations observed after 2 years of age, most could be classified into three types; classical (tolerate less than 600 mg of leucine per day, N=2), intermediate (N=3) and intermittent (N=3) types. In the other patient two of these three phenotypes were present. The BCKDH activities measured at a lower α-ketoisovaleric acid concentration (0.054 mM) were 0.026±0.015 in classical, 0.118±0.016 in intermediate and 0.625±0.139 in intermittent types and 7.052±0.779 (nmol/h per milligram of protein) in two controls, respectively; the differences being statistically significant (P<0.01, classical vs intermediate types; P<0.01, intermediate vs intermittent types; P<0.01, intermittent vs control). Kinetic and immunochemical analyses of the BCKDH revealed that, although there are a few exceptions, classical, intermediate and intermittent types correspond to the enzyme properties of sigmoidal kinetics with E1β subunit deficiency, near-sigmoidal kinetics with E1β subunit deficiency and hyperbolic kinetics with E2 subunit deficiency of the BCKDH, respectively.

Molecular diagnosis of maple syrup urine disease : screening and identification of gene mutations in the branched-chain α-ketoacid dehydrogenase multienzyme complex

Maple syrup urine disease (MSUD), an autosomal recessive inborn error of metabolism, is due to defective oxidative decarboxylation of the branched-chain 7-ketoacids (BCKAs) derived from transamination of the three branched-chain amino acids, valine, leucine and isoleucine. MSUD is a heterogeneous disorder and classical, intermittent, intermediate and thiamine-responsive phenotypes have been identified (Danner and Elsas 1989). The oxidative decarboxylation of three BCKAs is catalysed by the branchedchain e-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial macromolecular multienzyme complex consisting of the three catalytic components: the branched-chain ~-ketoacid decarboxylase (El), the dihydrolipoyltransacylase (E/) and dihydrolipoamide dehydrogenase (E3). The E t component is further composed of two subunits, E 1~ and Ex ft. The E t and E2 components are specific for BCKDH, whereas E 3 is identical to that associated with pyruvate dehydrogenase and ~ketoglutarate dehydrogenase complexes. In addition, the BCKDH complex contains two regulatory enzymes, a specific kinase and a specific phosphatase, that control the activity of the BCKDH complex through a phosphorylation-dephosphorylation cycle (Danner and Elsas 1989; Yeaman 1989). Thus, there are at least six genetic loci encoding this multienzyme complex and a mutation in any of these loci can result in dysfunction of the BCKDH complex. To clarify mechanisms involved in MSUD, measurements of the enzyme activity in cultured cells (Dancis et al 1972; Jinno et al 1984a), measurements of the generation time in cultured cells (Jinno et al 1984a), complementation analysis (Lyons et al 1973; Jinno et al 1984b) and immunoblot analysis (Danner et al 1985; Indo et al 1987, 1988; Fisher et al 1989) have been done, To further elucidate the molecular mechanisms of MSUD, we and others isolated and characterized cDNAs encoding BCKDH-E 1 a (Zhang et al 1987, 1988; Fisher et al 1989), E1 fl (Nobukuni et al 1990a, b), E2 (Hummel et al 1988; Danner et al 1989; Nobukuni et al 1989) and E 3 (Otulakowski and

Maple syrup urine disease : clinical and biochemical significance of gene analysis

The BCKDH complex consists of three catalytic components; branched chain c~ketoacid decarboxylase (El), dihydrotipoyl transacylase (E2), and dihydrolipoamide dehydrogenase (E3). E1 is further composed of two subunits, EV~ and E~/3. E~ and E 2 components are specific to BCKDH. The E3 component is common among the three ketoacid dehydrogenase complexes, BCKDH, pyruvate dehydrogenase and eketoglutarate dehydrogenase complexes. The BCKDH complex also contains two specific regulatory enzymes, a kinase and a phosphatase, compounds that are responsible for regulating the catalytic activity through phosphorylation and dephosphorylation. EIa is the catalytic subunit phosphorylated at two serine residues and is responsible for regulating the catalytic activity, by covalent modification. E 2 catalyses transfer of the acyl group from the lipoyl moiety to coenzyme A and forms the structural core of the enzyme complex and to this, El, E3, kinase and phosphatase are bound through non-covalent interactions. The function of E1/3 is unknown (Danner and Elsas t989; Yeaman, 1989). Impaired BCKDH activity leads to maple syrup urine disease (MSUD), an autosomal recessive inborn error of metabolism. MSUD is a heterogeneous disorder and the several different phenotypes heretofore identified include classical, intermediate, intermittent, and thiamine-responsive types as based on the clinical features (Danner and Elsas, 1989). To elucidate the molecular mechanisms of MSUD, we and other investigators isolated and characterized cDNAs encoding human BCKDH EV~ (Zhang et al., 1988; Fisher et al., 1989), E1/3 (Nobukuni et al., 1990), and E 2 (Hummel et at., 1988; Danner et al., 1989; Nobukuni et al., 1989).

Neonatal onset of medium-chain Acyl-CoA dehydrogenase deficiency in two siblings

Two male siblings with medium-chain acyl-CoA dehydrogenase deficiency were reported, in whom the enzyme activity was essentially undetectable and the symptoms and signs, including cyanosis, apnea, low body temperature, hypoglycemia and hyperammonemia, appeared within 48 hours of life. Muscle weakness and cardiomegaly in association with morphological abnormalities of mitochondria in skeletal and cardiac muscles, respectively, were found on electron microscopic examination in one of them. These observations suggest that the patients suffered from the most severe form of the disease, which has not been described in the literature.