Isolation and characterization of mutations in the human holocarboxylase synthetase cDNA.

Holocarboxylase synthetase (HCS) plays an essential role in biotin utilization in eukaryotic cells and its deficiency causes biotin-responsive multiple carboxylase deficiency in humans. We have cloned the human HCS cDNA and show that antiserum against the recombinant protein immunoprecipitates human HCS. A one base deletion resulting in a premature termination and a missense mutation (Leu to Pro) were found in cells from siblings with HCS deficiency. Human HCS shows homology to BirA, which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. The human HCS gene maps to chromosome 21q22.1.

Identification of a common mutation in Finnish patients with nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is an autosomal recessive metabolic disorder caused by the defects in the glycine cleavage system (GCS; EC 2.1.2.10), a multienzyme system that consists of four individual components. NKH is a rare disorder in many countries, but with a very high incidence in northern Finland. To understand the genetic background of this high incidence, we examined the GCS in a typical case of NKH at the molecular level. The activity of P protein, a component of the GCS, was not detected in the lymphoblasts of the patient, while P protein mRNA of a normal size and level was present in the cells. Structural analysis of P protein mRNA from the patient revealed a single nucleotide substitution from G to T in the protein coding region, which resulted in an amino acid alteration from Ser564 to Ile564. No P protein activity was detected when the mutant P protein with this amino acid substitution was expressed in COS 7 cells. The patient was homozygous for this mutation. Furthermore, this mutation was present in 70% (14 of 20) of P protein gene alleles in Finnish patients with NKH, whereas it was not found in 20 alleles of non-Finnish patients. The results suggest that this mutation is responsible for the high incidence of NKH in Finland.

Deficiency of 3-methylglutaconyl-coenzyme A hydratase in two siblings with 3-methylglutaconic aciduria.

We studied two patients with 3-methylglutaconic aciduria in order to determine the molecular defect. A new assay for 3-methylglutaconyl-coenzyme A (CoA) hydratase has been developed in which the substrate, [5-14C]3-methylglutaconyl-CoA, was synthesized using 3-methylcrotonyl-CoA carboxylase purified from bovine kidney. In this assay the products of the reaction are isolated by reverse-phase high performance liquid chromatography and the rates of conversion from substrate are measured. The Michaelis constant for 3-methylglutaconyl-CoA in normal fibroblasts was 6.9 mumol/liter. The mean activity of 3-methylglutaconyl-CoA hydratase in control fibroblasts was 495 pmol/min per mg protein. In the two patients the values were 11 and 17 pmol/min per mg protein, or 2-3% of normal.

Folate distribution in cultured human cells. Studies on 5,10-CH2-H4PteGlu reductase deficiency.

We have studied the distribution of folate coenzyme forms in cultured human fibroblasts from control lines and from lines derived from nine patients representing all of the published reports of 5,10-CH(2)-H(4)PteGlu reductase deficiency. Based on mobility on DEAE-Sephadex and differential microbiological assay the major folate fractions in extracts of human fibroblasts were 5-CH(3)-H(4)PteGlu, 10-CHO-H(4)PteGlu, and 5-CHO-H(4)PteGlu with smaller fractions, which included 5-CH(3)-H(2)PteGlu, 10-CHO-PteGlu, and H(4)PteGlu. Evidence that the 5-CHO-H(4)PteGlu may have been derived from 5,10-CH=H(4)PteGlu during extraction is presented. In most of the mutant fibroblasts the absolute concentration of 5-CH(3)-H(4)PteGlu was lower than in control cells but the proportion of intracellular folate which was 5-CH(3)-H(4)PteGlu was strikingly lower in mutant cells when determined by chromatography or differential microbiological assay. In both control and mutant cells most of the 5-CH(3)-H(4)-PteGlu was polyglutamate. The proportion of intracellular folate which was polyglutamate was similar in control and mutant cells. A direct relationship was observed between the proportion of cellular folate which was 5-CH(3)-H(4)PteGlu, and both the clinical severity of this disorder and the residual enzyme activity indicating that the distribution of different folates may be an important control of intracellular folate metabolism. These studies indicate that 5,10-CH(2)-H(4)PteGlu reductase is the only significant intracellular pathway for the generation of 5-CH(3)-H(4)PteGlu, that the activity of this enzyme regulates the level of this folate in control and mutant cells under conditions of culture used here, that the majority of intracellular folate is in the polyglutamate form, and that the relative distribution of folates may control folate metabolism by interaction in the various folate reactions.

Identification of holocarboxylase synthetase (HCS) proteins in human placenta.

Holocarboxylase synthetase (HCS) is a key enzyme in biotin utilization in eukaryotic cells. In a previous work from our laboratory, we described the cloning and sequencing of a full-length human HCS cDNA. Due to the presence of three candidate sites for initiation of translation, the identification of full-length HCS proteins remains uncertain. Using antibodies directed against human HCS sequences, we have identified, in human placenta, three cytosolic HCS proteins, of 86, 82 and 76 kDa. Similar results were observed in lysates of cells transfected with an HCS expression vector, as well as with human HCS cDNA transcribed and translated in a cell-free system. When anti-HCS antibodies were tested for their ability to inhibit HCS enzymatic activity, only the antibody directed against a region of HCS from Ile128 to Pro398, and not the antibodies against more proximal N-terminal regions inhibited HCS activity, suggesting that the sequence from Ile128 to Pro398 is essential for the catalytic activity of this enzyme. HCS synthesized in a cell-free system was not translocated into rat liver mitochondria. These results suggest that our human HCS cDNA encodes the cytosolic forms of the enzyme. These results also suggest that mRNA encoding cytosolic HCS can be translated from all three translation initiation codons, Met1, Met7 and Met58.

Molecular analysis of holocarboxylase synthetase deficiency: a missense mutation and a single base deletion are predominant in Japanese patients.

Holocarboxylase synthetase (HCS) deficiency is an inherited disease of biotin metabolism characterized by a unique pattern of organic aciduria, metabolic acidosis, and skin lesions. By analysis of five patients in four unrelated families, two mutations were identified: a transition from T to C which causes an amino-acid substitution of proline for leucine at position 237 (L237P) and a single deletion of guanine (delG1067) followed by premature termination. One patient was homozygous for the L237P mutation, three patients in two families were compound heterozygotes of the missense and deletion alleles, and the other patient was heterozygous for the L237P mutation. Inheritance was successfully demonstrated in all of the patients' families by a modified PCR followed by restriction enzyme digestion. The two mutations accounted for seven of eight mutant alleles, while neither mutation was detected in 108 normal healthy Japanese children (216 alleles). Transient expression in cultured fibroblasts from a patient showed that the L237P mutation was responsible for decreased HCS activity. These results suggest that the L237P and delG1067 mutations are frequent disease-causing mutations in Japanese patients with HCS deficiency. This PCR-based technique may therefore be useful for detecting mutations among Japanese patients.

Effects of Pathogen Density, Soil Moisture, and Soil pH on Biological Control of Clubroot in Chinese Cabbage by Heteroconium chaetospira.

The effects of soil moisture and pH, and pathogen resting spore density, on the effectiveness of the biological control of clubroot by the fungal endophyte Heteroconium chaetospira was evaluated in greenhouse and field experiments. Conditions favoring disease development included low pH (5.5) and high soil moisture content (80%), with significant reductions in the disease being observed at a higher pH (6.3 and 7.2) and lower soil moisture content (40 and 60%). In greenhouse tests, H. chaetospira effectively controlled clubroot (reducing the disease by 90 to 100%) at pathogen resting spore densities of 104 and 105 spores/g of soil at all soil pHs tested (5.5, 6.3, and 7.2). However, when the resting spore density was 106 spores/g of soil, plants were severely diseased, regardless of treatment, and H. chaetospira had no effect on disease. At a soil moisture content of 40%, disease occurrence was low, regardless of pathogen spore density, but disease was significantly lower in H. chaetospira-treated plants at pathogen spore density of 105 spores/g of soil. At 60% soil moisture content, H. chaetospira significantly could affect at pathogen spore densities of 104 and 105 but not 104/g of soil. At 80% soil moisture content, there was no effect of H. chaetospira at pathogen density. In situ, the soil moisture contents were constantly adjusted to relatively low to moderate (pF 2.2 to 2.4 and pF 2.0 to 2.2) and high (pF 1.6 to 1.8). Other environmental conditions, such as resting spore density and soil pH, were maintained at constant levels. Control plants (not treated with H. chaetospira) showed uniformly high disease levels and proportions of diseased plants across all three moisture treatments (disease index = 72 to 80, proportion of diseased plants 85 to 97%). In the field, H. chaetospira-treated plants at low soil moisture (pF 2.2 to 2.4, plot 1) had 68% disease reduction compared with untreated controls and 49% reduction at moderate moisture pF (pF 2.0 to 2.2, plot 2). There was no effect on disease by H. chaetospira at high soil moisture (pF 1.6 to 1.8, plot 3). Based on our results, H. chaetospira is an effective biocontrol agent against clubroot in Chinese cabbage at a low to moderate soil moisture range and a pathogen resting spore density of 105 (or lower resting spores per gram of soil in situ.

Association of a mutation in thiazide-sensitive Na-Cl cotransporter with familial Gitelman's syndrome.

Gitelman's syndrome is a variant of Bartter's syndrome, characterized by hypokalemia, hypomagnesemia, hypocalciuria, and hypovolemia. We have observed familial cases of Gitelman's syndrome, and a possible mutation in thiazide-sensitive Na-Cl cotransporter was investigated in this kindred. The proband was a 47-yr-old Japanese female, and her mother was also affected. Her parents and maternal grandparents are consanguineous. By using PCR-amplification and direct sequencing, we identified a novel non-conservative missense mutation at 623 amino acid position, which substitutes proline for leucine (L623P), and also creates an Nci I restriction site in the exon 15. The mutation was not detected in normal healthy subjects (n = 102). Nci I digestion of PCR-amplified exon 15 DNA fragments from individuals in the family indicated the autosomal recessive inheritance of the disorder. In conclusion, the L623P mutation in the thiazide-sensitive Na-Cl cotransporter gene is suggested to impair the transporter activity, and to underlie this familial Gitelman's syndrome; Gitelman's syndrome observed in this kindred has been inherited in an autosomal recessive fashion.

Effects of valproate on biogenesis and function of liver mitochondria.

The mitochondrial protein content of liver increased, and mitochondria enlarged after prolonged administration of valproic acid (VPA: N-dipropylacetic acid) to rats. The mitochondria showed low specific activities of membrane-bound enzymes, decreased content of cytochrome aa3, and decreased respiration in states 3 and 4. In vitro studies of amino acid incorporation suggested a decrease in protein synthesis in liver mitochondria from VPA-treated rats. Prolonged administration of VPA seems to impair mitochondrial structure and function.

Hyperphenylalaninemia due to dihydropteridine reductase deficiency: diagnosis by enzyme assays on dried blood spots.

Enzymatic diagnosis of hyperphenylalaninemia due to a deficiency of dihydropteridine reductase (DHPR) has previously been made by assay on liver biopsy samples, cultured skin fibroblasts, cultured lymphoid cell lines, or peripheral leukocytes. These procedures have some disadvantages for the purpose of early diagnosis of the disease. A simple method of DHPR assay using erythrocytes or dried blood spots on filter papers is described. The mean DHPR activity erythrocytes of control subjects was 3.20 +/- 0.70 (SD) nmoles/min/mg of hemoglobin, those of two patients were undetectable, and those of obligate heterozygotes for DHPR deficiency were approximately 50% of the mean control value. The assay on erythrocytes required only a 5-microliters volume of whole blood for one test. The DHPR activities in dried blood spots on filter papers from 100 normal newborns were 5.77 +/- 1.16 nmoles/min per 5-mm diameter disc; those from normal older infants, children, and adults were 3.37 +/- 0.72 nmoles/min per disc; and those from two adolescent patients with DHPR deficiency were undetectable. No false-positive results were obtained. The stability of DHPR in dried blood on filter papers was enough to mail samples in an ordinary form to a specialist laboratory. The DHPR assay on erythrocytes of dried blood spots can be easily applied to all newborn infants with hyperphenylalaninemia detected using the Guthrie tests, and will facilitate the quick and confirmative detection of DHPR deficiency among them.

Retrospective survey of urea cycle disorders: Part 1. Clinical and laboratory observations of thirty-two Japanese male patients with ornithine transcarbamylase deficiency.

In a retrospective survey done from 1978-1988 in Japan, 32 male patients with ornithine transcarbamylase (OTC) deficiency were identified. We classified a neonatal and 2 late-onset groups, depending on clinical manifestations and the age at onset; group 1 (0-28 days; N = 10), group 2 (29 days-5 years; N = 13), and group 3 (greater than 5 years; N = 9). Compared to findings in the group 2 patients, there was a higher rate of mortality and a higher incidence of mental retardation in association with a great decrease in enzyme activity in group 1. In group 3, the mortality rate and enzyme activities were similar to those in group 1. However, patients in this group were asymptomatic prior to the first episode. Enzyme activities were measured mostly in autopsy samples. The serum citrulline levels (enzyme product) were highest in this group. Thus, the mutant enzymes were apparently labile with greater activities in vivo than in vitro. Treatments, including a protein-restricted diet, arginine supplementation, and sodium benzoate administration, resulted in a favorable prognosis for survivors with partial enzyme deficiency. We wish to emphasize that the incidence of late onset of this disease is higher than heretofore considered.

Retrospective survey of urea cycle disorders: Part 2. Neurological outcome in forty-nine Japanese patients with urea cycle enzymopathies.

We analyzed neurological data, including DQ or IQ, EEG, and CT scan, in 49 patients with urea cycle enzymopathies, all of whom were included in a retrospective survey from 1978-1988 in Japan. We classified 3 groups depending on age-at-onset: group 1 (0-28 days, N = 11), group 2 (29 days-5 years, N = 31), and group 3 (greater than 5 years, N = 7). The least DQ or IQ score and the highest CT score, representing the most severe brain damage was found in group 1, and the highest DQ or IQ and the least CT score was found in group 3. Intermediate scores of both parameters were found in group 2. There was a negative correlation between these 2 parameters (r = -0.82, P less than 0.01). Abnormal EEG during the attack-free period was predominantly observed in patients with CT abnormalities compared to those with a normal CT scan (P less than 0.01). Approximately 40% of the patients, mostly in groups 2 and 3 (92.8%) had normal findings in all 3 parameters. Thus, the magnitude of developmental abnormalities is clearly related to the degree of brain damage and to the age-at-onset of these diseases.

Novel mutations in the connexin 26 gene (GJB2) responsible for childhood deafness in the Japanese population.

Mutations in the connexin 26 gene (GJB2), which encodes a gap-junction protein and is expressed in the inner ear, have been shown to be responsible for a major part of nonsyndromic hereditary prelingual (early-childhood) deafness in Caucasians. We have sequenced the GJB2 gene in 39 Japanese patients with prelingual deafness (group 1), 39 Japanese patients with postlingual progressive sensorineural hearing loss (group 2), and 63 Japanese individuals with normal hearing (group 3). Three novel mutations were identified in group 1: a single nucleotide deletion (235delC), a 16-bp deletion (176-191 del (16)), and a nonsense mutation (Y136X) in five unrelated patients. The 235delC mutation was most frequently observed, accounting for seven alleles in 10 mutant alleles. Screening of 203 unrelated normal individuals for the three mutations indicated that the carrier frequency of the 235delC mutation was 2/203 in the Japanese population. No mutation was found in group-2 patients. We also identified two novel polymorphisms (E114G and I203T) as well as two previously reported polymorphisms (V27I andV37I). Genotyping with these four polymorphisms allowed normal Japanese alleles to be classified into seven haplotypes. All 235delC mutant alleles identified in four patients resided only on haplotype type 1. These findings indicate that GJB2 mutations are also responsible for prelingual deafness in Japan.

Glycogen storage disease type Ib: structural and mutational analysis of the microsomal glucose-6-phosphate transporter gene.

Glycogen storage disease type Ib is caused by a mutation in the gene encoding microsomal glucose-6-phosphate (G6P) transporter. We determined the exon/intron organization of the G6P transporter gene. Four overlapping genomic fragments containing the entire coding region of the gene were amplified by polymerase chain reaction (PCR) using exonic primers, and their nucleotide sequences were determined. The gene spans 4.5 kb and has eight exons. All exon/intron boundaries adhered to the canonical AG/GT rule. We then designed eight pairs of PCR primers to amplify all coding exons for a mutational analysis and studied five Japanese patients with the disease. Two novel homozygous mutations were identified in two families: a three-base deletion (delV235) in exon 2 in a consanguineous family and a splicing mutation (IVS7+1G-->T) in intron 7 in a nonconsanguineous family. Patient 3 was a compound heterozygote of W118R and IVS1+1G-->A, both of which we previously identified [Kure et al., 1998: Biochem Biophys Res Commun 248:426-431]. Patients 4 and 5 were homozygotes of W118R. Including our previous study, we found a total of ten W118R alleles in nine Japanese patients. The results support our previous suggestion that W118R is prevalent among Japanese patients. The genomic sequence data and mutation spectrum obtained from the Japanese patients will facilitate genetic diagnosis of glycogen storage disease type Ib.

Heterogeneous mutations in the glucose-6-phosphatase gene in Japanese patients with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by glucose-6-phosphatase (G6Pase) deficiency. It is characterized by short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. Various mutations have been reported in the G6Pase gene (G6PC). However, in Japanese patients, a g727t substitution was found to be the major cause of GSD-Ia, accounting for 20 of 22 mutant alleles [Kajihara et al., 1995], and no other mutations have been found in this population. We analyzed four Japanese GSD-Ia patients and identified three other mutations in addition to the g727t. They included two missense mutations (R83H and P257L) and one nonsense mutation (R170X). Each of the three mutations exhibited markedly decreased G6Pase activity when expressed in COS7 cells. A patient homozygous for R170X showed multiple episodes of profound hypoglycemia associated with convulsions, while P257L was associated with a mild clinical phenotype. The presence of R170X in three unrelated families may implicate that it is another important mutation in the etiology of GSD-Ia in Japanese patients. Thus, the detection of non-g727t mutations is also important in establishing the DNA-based diagnosis of GSD-Ia in this population.

Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of glucose-6-phosphatase (G6Pase) that is expressed in the liver, kidney, and intestinal mucosa. Clinical manifestations include short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. To elucidate a spectrum of the G6Pase gene mutations and their frequencies, we analyzed mutations in 51 unrelated Japanese patients with GSD-Ia. The most prevalent mutation was g727t, accounting for 88 of 102 mutant alleles examined, followed by R170X mutation, which accounted for 6 mutant alleles, and R83H mutation which was observed in 3 mutant alleles. In addition, 3 different, novel mutations, IVS1-1g

Glycogen storage disease confined to the heart with deficient activity of cardiac phosphorylase kinase: a new type of glycogen storage disease.

The case of a male infant with marked deposition of glycogen, confined to the heart, is presented. Clinically, prominent cardiomegaly had been evident from immediately after birth until the infant's death due to heart failure. There were no significant clinical manifestations in other organs, including liver and skeletal muscle, during the clinical course. Autopsy revealed abnormal deposition of normally structured glycogen in the heart, but no deposition in the liver, skeletal muscle, or other systemic organs. This unusual pattern of glycogen deposition was also confirmed by measurement of the glycogen content of each organ. This is the first report of glycogen storage disease confined to the heart. Enzymatic analysis revealed no decrease in the activities of acid maltase, amylo-1,6-glucosidase, and phosphorylase in the heart or in the liver or skeletal muscle. However, phosphorylase kinase activity was not detectable in the heart, although high activity levels were observed in the liver and skeletal muscle. In this case the inborn error of metabolism responsible for the isolated deposition of glycogen in heart muscle may have been due to a deficiency of cardiac phosphorylase kinase.

Ocular findings in childhood lactic acidosis.

We examined ophthalmologically nine children with lactic acidosis. All showed abnormal ocular findings. Optic atrophy was present in six patients, nystagmus in three, blepharoptosis in one, cataract in one, and limitation on abduction in one. We believe that optic atrophy is the most frequent ocular finding and that nystagmus and ophthalmoplegia are common associations in patients with childhood lactic acidosis.

Endonuclease activation following focal ischemic injury in the rat brain.

The structural changes which occur in chromatin DNA after ischemic brain injury are poorly understood. This study examined the appearance of double-strand DNA breaks and the temporal profile of DNA degradation following focal ischemic injury in rat brain. Focal cerebral ischemia was produced by tandem occlusion of the common carotid and proximal middle cerebral arteries. The effects of decapitation ischemia were also studied by DNA analysis. DNA was extracted by standard methods from the ischemic brain tissues and electrophoresed on a 1.5% agarose gel. With decapitation ischemia, random DNA cleavage was observed as a dense "smear" on the gel electrophoresis beginning 6 h after the ischemic insult, and increasing in amount thereafter. Focal ischemia provided DNA fragmentation, which is specific DNA cleavage at the internucleosomal linker regions, particularly in the caudoputamen. Coexisting random degradation and specific fragmentation of DNA was observed in the cortex following focal ischemia. To determine whether an endonuclease responsible for DNA fragmentation was present, nuclear proteins were extracted from normal brain nuclei and the endonuclease activity was determined using plasmid DNA and a nuclear incubation system. This demonstrated that brain nuclear proteins have Ca(2+)-dependent endonuclease activity which is related to DNA fragmentation. Ischemic injury causes both random and specific DNA cleavage in the brain, which is probably mediated by Ca(2+)-dependent endonuclease.