Human δ-aminolevulinate dehydratase: chromosomal localization to 9q34 by in situ hybridization

Summary The structural gene for human -aminolevulinate dehydratase (ALA-D) has been localized to chromosomal region 9q34 by in situ hybridization using a [¹²⁵I]-labeled human -aminolevulinate dehydratase cDNA. Of the 150 silver grains analyzed, 25% were localized to chromosome 9q, while 12% and 8% were on chromosomes 1p and 13q, respectively. The single chromosomal region q34 had over 90% of the total grains observed on chromosome 9. In contrast, the grains on chromosomes 1p and 13q were dispersed, consistent with the absence of any human ALD-D pseudogenes. Southern blot analysis of somatic cell hybrids informative for ALA-D (Wang et al. 1985) also was consistent and supported the finding of only one locus for this heme biosynthetic enzyme.     

ALA-dehydratase activity inZea mays L.

Summary The presence and activity of -aminolevulinic acid dehydratase (ALA-D, E.C.4.2.1.24) inZea mays L. was studied. Seedlings of several inbred lines and their hybrids were employed as enzyme source, and enzyme activity and the kinetic parameters (V_max and K_M) determined. The wide variations of the enzyme activity and kinetic parameters found in the tested lines support the hypothesis that ALA-D varies in concentration and isoenzyme distribution. Enzyme activity was greater in some hybrids than in their parent lines. As the same hybrids correspond to varieties appreciated for their high productivity and good morphological characteristics it is suggested that ALA-D activity levels could be adopted as an auxiliary parameter for establishing heterosis.     

Familial lecithin-cholesterol acyltransferase: Identification of heterozygotes with half-normal enzyme activity and mass

Summary Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Canadian kindred of Italian and Swedish descent with familial LCAT deficiency. Four subjects had LCAT mass of 5.21±0.87 g/ml (mean±SD) and LCAT activity of 98.8±12.0 nmol/h/ml, well within their respective normal ranges. Five family members, including the parents, the maternal grandmother, and two of four siblings of the LCAT deficient subjects, had enzyme mass (2.85±0.32 g/ml) and activity (50.8±6.3 nmol/h/ml) approximately one-half that of normal levels. These presumed heterozygotes had normal levels of apolipoproteins A-I, A-II, B and D. The two subjects with LCAT deficiency had no detectable LCAT mass (below 0.1 g/ml) or LCAT activity (below 0.76 nmol/h/ml), apolipoprotein A-I and D levels approximately 50% of normal, and apolipoproteins B and A-II levels only 30–35% of normal. LCAT deficiency in this family is determined by an autosomal recessive mode. Furthermore, LCAT levels and activity are determined by two autosomal codominant alleles, LCATⁿ, the normal LCAT gene, and LCAT^d, the LCAT deficiency gene.     

β-Galactosidase-neuraminidase deficiency in adults: Deficiency of a freeze-labile neuraminidase in leukocytes and fibroblasts

Summary 4-Methylumbelliferyl neuraminidase activity was studied in fibroblasts, leukocytes, and frozen tissues from adult patients with -galactosidase-neuraminidase deficiency and specific clinical manifestations. This enzyme was almost completely deficient in fibroblasts, but the residual activity was relatively high (20% of the control mean) in the leukocytes from the patients. The frozen liver from one patient showed the enzyme activity as high as controls.This enzyme consisted of two components, freeze-labile and freeze-stable, and it was demonstrated that only the labile enzyme was deficient in fibroblasts and leukocytes. The apparently normal activity of neuraminidase in frozen autopsy tissues of a patient may be explained by the loss of the labile component in control tissues after a long-term freezing. The neuraminidase activity was variable in parents and no definite conclusion was drawn on the hereditary nature of the disease.     

Familial lecithin-cholesterol acyltransferase deficiency in a Japanese family: Evidence for functionally defective enzyme in homozygotes and obligate heterozygotes

Summary Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Japanese family with familial LCAT deficiency. The two LCAT-deficient subjects had LCAT mass approximately 40–46% of normal (2.65 and 2.31 g/ml respectively, as compared with normal levels of 5.76±0.95 g/ml in 19 Japanese subjects) and enzyme activity less than 10% of normal (9.1 and 8.3 nmol/h/ml respectively, as compared with normal levels of 100 nmol/h/ml). All obligate heterozygotes examined, including the father of the two LCAT-deficient subjects, and all five children of the deficient subjects had LCAT mass approximately 72–80% of the normal LCAT mass (4.12, 4.38, 4.45, 4.48, 4.49, 4.61 g/ml, respectively) and LCAT activity approximately half normal (51.9, 52.4, 54.2, 56.6, and 57.2 nmol/h/ml). We conclude that the two LCAT-deficient subjects of this family have functionally defective enzyme. Furthermore, the data suggest that the plasma of the obligate heterozygotes contain both normal and functionally defective enzymes.     

GD (-) Aachen,a new variant of deficient glucose-6-phosphate dehydrogenase

Summary A deficient G-6PD variant was discovered in 4 males of one family from north-western Germany. Five generations of this family could be studied.The deficient G-6PD was a new variant, called Gd (-) Aachen. Its main characteristics are the following: severe enzyme deficiency in erythrocytes (3% of normal), contrasting with an almost normal activity in leukocytes; normal molecular specific activity (i.e., normal ratio enzyme activity/cross-reacting material); slow mobility in starch gel electrophoresis (92–94% of normal); increased Michaelis constant for glucose-6-phosphate (60–70 M) and NADP⁺ (20–25 M); decreased inhibition constant by NADPH with respect to NADP⁺ (7 M); increased inhibition by ATP; normal utilization of the substrate analogues; slightly biphasic pH curve; thermal instability, and normal activation energy of the enzymatic reaction.The relationships between the hematologic disorders (severe and frequent hemolytic crises) and the unfavorable kinetic modifications are discussed.with the technical assistance of Joelle Marie and Dominique CottreauDedicated to Prof. Dr. H. Schonenberg, Aachen, on his 60th birthday. The first results of this work were presented in part at the Kongress der Deutschen Kinderärzte, München.     

Glucose-6-phosphate dehydrogenase in the population of northern Thailand: Evidence for two common electrophoretic variants with deficient enzyme activity

Summary Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, identified by a dye decolorization test, was found in 101 (12.5 percent) of 811 male subjects from northern Tailand. Blood samples from 169 subjects with normal G-6-PD activity and from all 101 subjects with G-6-PD deficiency were examined by electrophoresis on cellulose acetate gel with the following results: In all samples with normal G-6-PD activity the enzyme had the electrophoretic mobility of type B G-6-PD. 73 of the 101 G-6-PD deficient samples had the same mobility and are therefore probably identical with the common Mediterranean variant B-. 16 of the 101 deficient samples contained an electrophoretically fast G-6-PD, and 1 sample a slow variant. In 11 deficient samples the enzyme could not be made visible. Kinetic studies on crude hemolysates suggest that the fast variant has a higher mean activity and heat stability in comparison to the B- variant.Established and supported by Stiftung Volkswagenwerk, Hannover.     

A two-year-old patient with an atypical expression of GM1-β-galactosidase deficiency: Biochemical,immunological, and cell genetic studies

Summary Cultured skin fibroblasts from a 2-year-old boy with an atypical form of -galactosidase deficiency have been studied. With the artificial substrate 4-methylumbelliferyl--D-galactopyranoside, 5–15% residual activity was found in fibroblasts from this patient. Most of this activity was in the monomeric A form of the enzyme, very little in the multimeric B form. Km value, pH profile, and heat lability of the mutant enzyme were similar to those of -galactosidase from control fibroblasts. Immunological studies showed that the mutant enzyme cross-reacted with an antiserum raised against human liver -galactosidase, but the catalytic activity per unit antigenic activity was lower than normal. It was demonstrated by somatic cell hybridization that the gene mutation in this patient is different from that in patients with type 1 or type 2 G_M1-gangliosidosis. No genetic complementation was found after fusion of fibroblasts from this patient with those from two other clinical variants of G_M1-gangliosidosis formerly designated type 3 and adult type 4.     

Demonstration of the spf-ash mutation in Spanish patients with ornithine transcarbamylase deficiency of moderate severity

We have found in patients with ornithine transcarbamylase (OTC) deficiency from two Spanish families (A and B), replacement by A of G at the 3-end of exon 4 of the OTC gene. The same mutation is found in the spf-ash mouse, a rodent model of mild OTC deficiency, causing a neutral R129H mutation and inefficient splicing at the 5donor site of the exon 4-intron 4 junction, with resultant 4%–7% residual OTC activity. The mutation, detected in our patients using polymerase chain reaction (PCR) amplification of the ten OTC exons, single strand conformation polymorphism (SSCP) analysis and direct sequencing of PCR-amplified exon 4, results in the loss of a unique MspI restriction site which can be used for rapid diagnosis. The mutation was transmitted by the mother in family A and arose de novo in the patient in family B. Residual OTC activity, determined in a male and a female patient, was 1.3% and 3.5% of normal, respectively. Despite this low activity, the surviving patients have developed normally.     

X-linked dominant inheritance of partial phosphorylase kinase deficiency in mice

A new mouse strain, the V strain, with a partial deficiency of phosphorylase kinase has been established. The deficiency is caused by an X-linked dominant gene (Phk ^c ). Muscle extracts of homozygous and heterozygous females and hemizygous males have about 25% of the activity found in extracts of normal (C3H/HeHan) mice. This dominant phosphorylase kinase deficiency of the new V strain is different from that of the I-strain mice with the X-linked recessive deficiency of skeletal muscle phosphorylase kinase. The muscle extracts of V-strain and normal mice contain the same phosphorylase phosphatase activity of about 1 U/mg. Heart and liver extracts from V mice contained about 50% and 66%, respectively, of the phosphorylase kinase activity compared to that found in the same organs from the normal mice. The glycogen content of the skeletal muscle of the V strain was normal, i.e., 0.9 mg/g. Phosphorylase kinase was purified from the skeletal muscle of the V strain by (a) hydrophobic chromatography on methylamine Sepharose, (b) ammonium sulfate precipitation, and (c) gel filtration of Sepharose 4B. The enzyme has a similar structure to the normal murine and rabbit skeletal muscle enzyme, except that the proportion of the subunits differs. The molar ratio of the subunits of the V strain mice is (+)::=0.54:1:1.169, in comparison with that of the rabbit (+)::=1.1:1.0:1.0 and that of normal murine enzyme 0.9:1.0:0.7.This work was supported by the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, West Germany and of the Fonds der Chemie, West Germany, and forms part of the md thesis of A. Vrbica.     

Evidence for the deficiency of β-glucosidase-activating factor in fibroblasts of patients with I-cell disease

Summary Reduced activity of -glucosidase was shown in the cultured skin fibroblasts of four patients with I-cell disease when the enzyme was tested without the use of detergents. In the presence of taurocholate and triton X100 -glucosidase activity was normal. This suggested a deficiency of a -glucosidase-activating factor in I-cell fibroblasts rather than of the enzyme itself. The deficiency of -glucosidase activity was corrected to some extent by mixing cell lysates, and more effectively by cocultivation and fusion of I-cell disease and Gaucher fibroblasts. These results present evidence for the presence of a -glucosidase-activating factor in normal and Gaucher fibroblasts. In fibroblasts of patients with I-cell disease this activator is probably deficient, as is the case for most lysosomal enzymes.     

Pyruvate dehydrogenase (PDH) deficiency caused by a 21-base pair insertion mutation in the Elα subunit

Summary We report the molecular characterization of a case of a functional PDH-El (E1 subunit of pyruvate dehydrogenase) deficiency, a cause of severe congenital lactic acidosis. Residual PDH-El activity was reduced to 10% of normal values, although the subunit appeared to be quantitatively and qualitatively normal at the protein level as determined by Western blotting. The sequence of PDH-El mRNA and the corresponding genomic DNA revealed an in-frame 21-bp insertion between codons 305 and 306 of the normal Ela cDNA. The mutational insert commences with a novel GAT codon and is a nearly perfect tandem duplication of the wild type DNA sequence. A serine phosphorylation site regulating the activity of the PDH complex is altered by this insertion, which in all likelihood is responsible for the functional enzymatic deficiency leading to lactic acidosis.     

First reaction of riboflavin biosynthesis —Catalysis by a guanosine triphosphate cyclohydrolase from yeast

An enzyme that uses GTP as a substrate for the formation of formate and a 2.4.5-triamino-6-hydroxypyrimidine derivative has been determined in the riboflavin overproducing yeast Pichia guilliermondii. In rib 1 mutants this enzyme is absent. This implies an involvement of the enzyme in the riboflavin biosynthesis and indicates that GTP is a purine precursor of riboflavin. Some properties of the GTP cyclohydrolase (substrate specificity, pH and temperature optima, activators and inhibitors, molecular weight, stability) were studied using a partly purified enzyme preparation. Cells grown under riboflavin overproducing conditions (iron deficiency) have 20–40-fold increased enzyme activity as compared with non-overproducing cultures (supplemented with iron).Non-Standard Abbreviations RF riboflavin - DHRiboyslAP £ 2.5-diamino-6-hydroxy-4-(1-d-ribosylamino)pyrimidine-5-phosphate - DHRibitylAP 2.5-diamino-6-hydroxy-4-(1-ribitylamino)pyrimidine - neopterin 6-(trihydroxypropyl)pterin - DMP 6.7-dimethylpterin - Fe iron deficient condition - +Fe supplemented with iron     

‘Gd(-) Hôtel Dieu’: A new G-6PD variant with chronic hemolysis in a Negro patient from Senegal

Summary A G-6PD deficiency was detected in a Negro patient from Senegal suffering from congenital nonspherocytic hemolytic anemia.The main characteristics of this variant were: profound defect of G-6PD activity in the red cells, decreased immunologic specific activity, fast electrophoretic mobility, decreased K_m-G-6P and normal K_m-NADP⁺, normal inhibition by ATP and NADPH, slightly increased utilization of the substrate analogues, slightly biphasic pH curve, high heat lability, subnormal activation energy.The characteristics of this variant being unique, it was called G-6PD Hôtel Dieu.     

B lymphocyte galactosyltransferase protein levels in normal individuals and in patients with rheumatoid arthritis

We have quantified the level of 4-galactosyltransferase protein in human B lymphocytes using an ELISA-based assay. Between 1-10ng of 4-galactosyltransferase was detected per mg total cellular protein, indicating that this enzyme constitutes <0.001% of B lymphocyte cellular protein. Akin to previous studies, individuals with rheumatoid arthritis exhibited reduced lymphocytic galactosyltransferase enzyme activity compared with normal controls when using ovalbumin as the acceptor substrate. The levels of enzyme protein present in B lymphocytes from patients with rheumatoid arthritis was, however, not reduced suggesting that the B lymphocyte galactosyltransferase catalytic activity may be regulated post-translationally.     

Relationship between α-L-fucosidase deficiency in plasma and α-L-fucosidase activity in leukocytes

Summary After testing a population sample of 185 hospitalized Italian children for the plasma -L-fucosidase deficiency and establishing an approximate threshold value between heterozygotes and wild-type homozygotes, we analyzed by two statistical methods the distribution of the two genotypes. The results obtained by probit analysis agree with threshold and average values expected on the basis of the Hardy-Weinberg equilibrium.In addition, the level of -fucosidase in leukocytes of 12 individuals with deficiency of -fucosidase in plasma was found to be significantly lower than that of 61 controls (P<0.005). These results indicate that the mutation(s) causing a deficiency of -fucosidase in plasma is (are) also expressed in leukocytes.     

On the molecular nature of the Duarte variant of galactose-1-phosphate uridyl transferase (GALT)

Galactosemia is an inborn error of galactose metabolism secondary to deficiency of galactose-1-phosphate uridyl transferase (GALT). GALT is a polymorphic enzyme and Duarte (D) is the most common enzyme variant. This variant is characterized by faster electrophoretic mobility and reduced activity. Duarte/galactosemia compound heterozygotes (D/G) are commonly identified in galactosemia newborn screening programs. However, these patients do not generally require treatment. By using a candidate mutation approach to define the molecular basis of the Duarte variant of GALT, a close association between the previously reported N314D polymorphism and the Duarte variant of GALT was found. We suggest that N314D encodes the D variant of GALT and that molecular testing for N314D might be useful to confirm a biochemical diagnosis of Duarte variant of GALT.     

Screening for Fabry disease in patients undergoing dialysis for chronic renal failure in Turkey: Identification of new case with novel mutation

Background

Chronic renal failure (CRF) is a serious complication of Fabry disease (FD). The aims of the present study were to determine the prevalence of unrecognized FD in Turkish hemodialysis population and to investigate the molecular background.

Method

Primarily, α-galactosidase A (α-Gal A) activity was investigated on DBS in 1136 patients of both sexes who underwent dialysis for CRF in Turkey. The disease was confirmed by analyzing enzyme activity in leukocyte and GLA gene sequencing in all patients in whom α-Gal A level was 40% of normal or less.

Results

Mean age of the patients (44.5% female, 52.5% male) was 56.46 ± 15.85 years. Enzyme activity was found low with DBS method in 12 patients (four males, eight females). Two men, but no women, were diagnosed with FD by enzymatic and molecular analysis. In consequence of genetic analysis of a case, a new mutation [hemizygote c.638C>T (p.P214S) missense mutation in exon 5] was identified, which was not described in literature. Family screening of cases identified six additional cases.

Conclusion

As a result of this initial screening study performed on hemodialysis patients for the first time with DBS method in Turkey, the prevalence of FD was detected as 0.17%. Although the prevalence seems to be low, screening studies are of great importance for detecting hidden cases as well as for identifying other effected family members.     

Inheritance of the enzyme deficiency in three neurolipidoses: variant 0 of Tay-Sachs disease (Sandhoff's disease), classic tay-sachs disease,and metachromatic leukodystrophy. Identification of the heterozygous carriers

Summary The recessive inheritance of the enzyme deficiencies (total hexosaminidase, hexosaminidase A, arylsulfatase A) in nine families with neurolipidoses (variant 0 of Tay-Sachs disease=Sandhoffs disease, classic Tay-Sachs disease, metachromatic leukodystrophy) has been studied. 62 family members were identified as: genotypically normals, phenotypically normal heterozygous carriers, or patients, according to the enzyme levels (normal, about half normal or low) in the leukocytes. The activities of the lysosomal enzymes involved in the diseases are related to the mean relative activity of three or four lysosomal reference enzymes thereby identifying the heterozygous carrier state by the enzyme level about half the normal enzyme. It cannot be completely excluded that the heterozygous state or total enzyme deficiency occur more frequently than would be expected according to Mendel's laws. This problem is discussed.

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Zusammenfassung Bei 9 Familien (62 untersuchten Personen) mit Neurolipidosen (Variante 0 der Tay-Sachsschen Erkrankung, klassische Tay-Sachssche Erkrankung, metachromatische Leukodystrophie) wird, der recessive Erbgang der Enzymdefekte (Total-Hexosaminidase, Hexosaminidase A, Arylsulfatase A) aufgezeigt. Die einzelnen Personen werden auf Grund der Enzymspiegel in den Leukocyten als genotypisch normal, heterozygot (ca. 50%ige Erniedrigung bei phänotypisch Normalen) oder krank (Enzymdefekt) identifiziert. —Werden die Werte der betroffenen lysosomalen Enzyme auf die durchschnittliche relative Aktivität von drei oder vier lysosomalen Referenzenzymen bezogen, so wird die Erniedrigung des Enzymspiegels bei Heterozygoten in fast jedem Fall erfaßt.—Es kann nicht völlig ausgeschlossen werden daß die Enzymdefekte etwas häufiger, als nach den Mendelschen Regeln anzunehmen ist, weitervererbt werden. Eine Hypothese dafür wird aufgestellt.

     

Genetic heterogeneity of propionic acidemia: Analysis of 15 Japanese patients

Summary Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. We have analyzed the molecular heterogeneity of Japanese propionic acidemia patients using anti-human PCC antiserum and cDNA clones coding for the two protein subunits ( and ) of the enzyme. The steady state levels of both and subunits of PCC from 15 Japanese patients were determined by Western blot. Three patients had neither nor subunits, and the amounts of both and subunits were low in 3 other patients. According to our previous data, we classified these 6 patients as having subunit deficiency. In the remaining 8 patients, subunits were normal, but the subunits were aberrant. Two patients had low levels of normal-sized subunits and 6 had subunits smaller than normal in size and greatly reduced in quantity. These 8 patients were assigned to the subunit deficiency category. One patient had apparently normal and subunits. We could not determine this patient's primary defect. These data reveal the genetic heterogeneity of molecular defects causing propionic acidemia in the Japanese. Southern blot analysis did not reveal any gross alteration in gene structure when DNA was digested withHindIII,EcoRI andTaqI. However, DNA from 3 -subunit-deficient patients, when digested withMspI and probed with PCC cDNA, revealed a unique 2.7-kb band not observed in blots of DNA from any other patient or 15 normal controls. We conclude that this alteredMspI restriction map is the result of a mutation in the subunit gene of these patients.