Demethylation of the hypoxia induction factor 1 binding site of GPX3 at excess blood ammonia in propionic acidemia.

OBJECTIVES: Elevated levels of metabolites such as ammonia and propionylcarnitine in propionic acidemia (PA) lead to an increased reactive oxygen species (ROS) production which could activate and stabilize the epigenetic regulated hypoxia-inducible factor-1α (HIF-1α). In order to evaluate the DNA methylation status of the HIF-1α binding site in PA, we investigated the antioxidant gluthatione peroxidase 3 gene (GPX3) promoter region. DESIGN AND METHODS: Using leukocyte DNA extracted from bloodspots collected 2-4 days after birth from diet free newborns, the cytosine phosphodiester bond guanine (CpG) dinucleotides of a HIF-1α binding site (CGTTTTTTACG) in the promoter region of GPX3 was retrospectively analysed. Patients included 7 PA. and 7 healthy controls (KO) respectively. RESULTS: A demethylated TGTTTTTTATG allele was detected in 3 PA patients with blood ammonia (NH3) concentrations of 500, 595, and 987 umol/L respectively; a demethylated/partial methylated TGTTTTTTAC/TG allele in 4 PA patients (2 PA with blood NH3 = 213, 271 umol/L respectively); a partial methylated C/TGTTTTTTAC/TG allele in 5 healthy controls respectively; a partial methylated/methylated C/TGTTTTTTACG allele in 2 healthy controls. CONCLUSION: Our results suggest that at excess NH3, the DNA methylation status of the HIF-1α binding site of GPX3 in newborns with PA is demethylated (TGTTTTTTATG allele). However, the demethylated allele has to be confirmed as a statistically significant change in more patients.

Concurrent FOXP3- and CTLA4-associated genetic predisposition and skewed X chromosome inactivation in an autoimmune disease-prone family.

CLTA4 is relevant for FOXP3(+)Treg cells, and the link between skewed X chromosome inactivation (XCI) and autoimmunity is recognized. The observation of immune dysregulation polyendocrinopathy enteropathy X-linked syndrome and multiorgan endocrine autoimmune phenomena in various members of one family, associated with a CTLA4 polymorphism and skewed XCI, provides an in vivo model of how mechanisms of immune dysregulation may cooperate.

Severe speech delay as the presenting symptom of guanidinoacetate methyltransferase deficiency.

Guanidinoacetate methyltransferase deficiency typically presents with muscular hypotonia, global developmental delay, extrapyramidal signs, and seizures during infancy and childhood. The authors report a 5-year-old child with guanidinoacetate methyltransferase deficiency who presented with severe speech delay, emphasizing the importance of an early screening for disorders of creatine synthesis and transport in every infant or child with isolated speech delay of unknown cause.

GAMT deficiency: features, treatment, and outcome in an inborn error of creatine synthesis.

BACKGROUND: Guanidinoactetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder of creatine synthesis. The authors analyzed clinical, biochemical, and molecular findings in 27 patients. METHODS: The authors collected data from questionnaires and literature reports. A score including degree of intellectual disability, epileptic seizures, and movement disorder was developed and used to classify clinical phenotype as severe, moderate, or mild. Score and biochemical data were assessed before and during treatment with oral creatine substitution alone or with additional dietary arginine restriction and ornithine supplementation. RESULTS: Intellectual disability, epileptic seizures, guanidinoacetate accumulation in body fluids, and deficiency of brain creatine were common in all 27 patients. Twelve patients had severe, 12 patients had moderate, and three patients had mild clinical phenotype. Twenty-one of 27 (78%) patients had severe intellectual disability (estimated IQ 20 to 34). There was no obvious correlation between severity of the clinical phenotype, guanidinoacetate accumulation in body fluids, and GAMT mutations. Treatment resulted in almost normalized cerebral creatine levels, reduced guanidinoacetate accumulation, and in improvement of epilepsy and movement disorder, whereas the degree of intellectual disability remained unchanged. CONCLUSION: Guanidinoactetate methyltransferase deficiency should be considered in patients with unexplained intellectual disability, and urinary guanidinoacetate should be determined as an initial diagnostic approach.

Use of denaturing HPLC to provide efficient detection of mutations causing guanidinoacetate methyltransferase deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive error of creatine synthesis characterized by cerebral creatine deficiency, accumulation of guanidinoacetate, mental retardation, epilepsy, and extrapyramidal symptoms. To date, 14 mutations of the GAMT gene in 27 patients have been reported. Mutation analysis was done using direct sequencing of PCR products and denaturing gradient gel electrophoresis in combination with direct sequencing. In contrast, we evaluated the efficiency of a newly developed DHPLC method to detect mutations in the GAMT gene by analysing DNA from 14 GAMT patients with known mutations. PCR amplification of both patient and control DNA was followed by formation of homoduplices and heteroduplices, and their detection by DHPLC. DHPLC identified all mutations tested and is the preferred choice of analytical method.

Lack of correlation between fatty acid oxidation disorders and haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome?

AIM: Fatty acid beta-oxidation defects comprise a heterogeneous group of disorders that may precipitate acute life threatening metabolic crises particularly during catabolic episodes. Several studies have demonstrated a possible association between fatty acid beta-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and severe pregnancy complications. However, the precise percentage of women with haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome associated with foetal fatty acid beta-oxidation defects is not known. METHODS: We carried out a multicentre retrospective study on 88 infants, born to women with HELLP syndrome. Acylcarnitine profiles from blood dried on filter paper cards were analysed by tandem mass spectrometry for the diagnosis of fatty acid beta-oxidation defects. In addition, we screened for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation using a standard restriction fragment length polymorphism polymerase chain reaction method. RESULTS: None of the infants studied carried the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation. There was no evidence of fatty acid beta-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, as expected by unremarkable acylcarnitine profiles, while three infants with fatty acid beta-oxidation defects were diagnosed in the control group. CONCLUSIONS: Neither foetal long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, including heterozygosity for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation, nor fatty acid beta-oxidation defects in general are a major risk factor for HELLP syndrome in Austria.

Characterization of seven novel mutations in seven patients with GAMT deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive error of creatine synthesis characterized by cerebral creatine deficiency, accumulation of guanidinoacetate, mental retardation, epilepsy and extrapyramidal signs. So far, six mutations have been identified in seven patients. We investigated seven new patients by screening the promoter, 3'UTR, and six exons and exon/intron boundaries using direct sequencing and denaturing gradient gel electrophoresis. The clinical and biochemical phenotype was characterized by scoring the degree of main clinical manifestations and by determination of urinary guanidinoacetate concentrations and of GAMT activity in fibroblasts / lymphoblasts, respectively. We identified 7 novel mutations, including c.64dupG (exon 1; 4/14 alleles); c.59G>C (exon 1; 3/14 alleles); c.491delG (exon 5; 2/14 alleles); c.160G>C (exon 1; 2/14 alleles); and c.152A>C (exon 1; 1/14 alleles); c.526dupG (exon 5; 1/14 alleles); c.521G>A (exon 5; 1/14 alleles), and two polymorphisms c.626C>T (exon 6) and c.459+71G>A (intron 4). Frameshift and missense mutations in exon 1 were prevalent in the 4 patients with the severe phenotype, however a clear genotype-phenotype correlation has not been established in the limited number of patients characterized so far.

[Evidence of osteocalcin expression in osteoblast cells of mandibular origin growing on biomaterials with RT-PCR and SDS-PAGE/Western blotting]. / Nachweis der Osteokalzinexpression osteoblastärer Zellen mandibulären Ursprungs, wachsend auf Biomaterialien, mittels RT-PCR und SDS-PAGE/Western Blotting.

A new approach to addressing difficult tissue reconstructive or replacement problems in the oral cavity is to engineer new tissue by using selective cell transplantation on polymer scaffolds. The current study characterized the osteoblastic nature of adherent mandibular cells on biomaterials, which could have a potential use as scaffolds for tissue engineering strategies. Cells of mandibular origin from one patient were cultivated on three different biomaterials (PepGen P-15 trade mark, Frios Algipore, and OsteoGraf/LD-700) for 7 and 14 days and osteocalcin expression was demonstrated by RT-PCR and SDS-PAGE/Western blotting. In order to explicitly characterize only the adherent cells on the biomaterials, we first separated the biomaterials with adherent cells from the culture plate before trypsinization. We could demonstrate that cell growth of adherent mandibular osteoblast-like cells was significantly higher on biomaterials with an organic component (PepGen P-15 trade mark ) in comparison to Frios Algipore and OsteoGraf/LD-700, respectively. In conclusion, only the explicit study of adherent cells at the gene and protein levels gives information about the osteoconductivity of biomaterials.

Arginine:glycine amidinotransferase deficiency: the third inborn error of creatine metabolism in humans.

Arginine:glycine amidinotransferase (AGAT) catalyzes the first step of creatine synthesis, resulting in the formation of guanidinoacetate, which is a substrate for creatine formation. In two female siblings with mental retardation who had brain creatine deficiency that was reversible by means of oral creatine supplementation and had low urinary guanidinoacetate concentrations, AGAT deficiency was identified as a new genetic defect in creatine metabolism. A homozygous G-A transition at nucleotide position 9297, converting a tryptophan codon (TGG) to a stop codon (TAG) at residue 149 (T149X), resulted in undetectable cDNA, as investigated by reverse-transcription PCR, as well as in undetectable AGAT activity, as investigated radiochemically in cultivated skin fibroblasts and in virus-transformed lymphoblasts of the patients. The parents were heterozygous for the mutant allele, with intermediate residual AGAT activities. Recognition and treatment with oral creatine supplements may prevent neurological sequelae in affected patients.

Molecular characterisation of 34 patients with biotinidase deficiency ascertained by newborn screening and family investigation.

This study characterises the spectrum of biotinidase mutations in 21 patients (17 families) with profound biotinidase deficiency (BD) and 13 unrelated patients with partial BD using a denaturing gradient gel electrophoretic mutation screening and selective sequencing approach. In 29 from 30 unrelated families we found biallelic mutations including four common mutations, D444H (frequency 23.3%), G98:d7i3(20.0%), Q456H(20.0%), T532M (15.0%) and nine rare mutations (V62M, R157H, A171T+D444H, C423W, D543H, L279W, N172S, V109G, 12236G-A) with frequencies less than 5.0%. Only three profound BD patients with G98:d7i3/G98:d7i3 and Q456H/Q456H genotypes and residual biotinidase activities of 0.0%, and 0.9% of normal activity developed clinical symptoms before biotin supplementation at 8 weeks of age. All other patients remained asymptomatic within the first months of life or even longer without treatment. Two patients homozygous for the frameshift mutation G98:d7i3 had no measurable residual enzyme activity. Twelve patients with partial BD had the D444H mutation in at least one allele. We conclude that, based on mutation analysis and biochemical examinations of the enzyme, it is currently not clearly predictable whether an untreated patient will develop symptoms or not, although it seems that patients with activities lower than 1% are at a high risk for developing symptoms of the disease early in life.

The genetic background modifies the effects of the obesity mutation, 'fatty', on apolipoprotein gene regulation in rat liver.

BACKGROUND: Obesity is associated with disorders of plasma lipid transport in many, but not in all obese subjects. The effects of obesity on the regulation of genes involved in plasma lipid transport may depend on specific mutations causing or contributing to obesity and/or on interactions of a specific obesity mutation with the genetic background. The 'fatty' (Glu269Pro) leptin receptor mutation causes severe obesity associated with hypertriglyceridaemia and altered hepatic apolipoprotein gene regulation in Zucker fatty rats. OBJECTIVE: To determine whether the effects of the obesity mutation 'fatty' on apolipoprotein gene regulation in rat liver depend on the genetic background. METHODS: We studied hepatic apolipoprotein (apo) A-IV, A-I, and C-III gene expression in obese rats carrying the 'fatty' mutation on the background of the Zucker or Wistar strain. RESULTS: Basal apoA-IV gene expression was increased in fatty rats of both strains, whereas apoA-I and apoC-III gene expression differed between Wistar and Zucker fatty rats: apoA-I gene transcription was reduced to half and apoC-III mRNA was increased two-fold in Wistar fatty, but not in Zucker fatty rats vs lean controls. A fish oil diet suppressed apoA-IV, but not apoA-I gene transcription in Wistar fatty rats, whereas in Zucker fatty rats apoA-IV transcription was unaffected, but apoA-I transcription was suppressed. CONCLUSIONS: Interactions of the 'fatty' leptin receptor mutation with the genetic background significantly affect the basal and diet-induced regulation of the apoA-IV, C-III and A-I genes in rat liver. The genetic background may therefore be a major determinant of the consequences of a specific obesity mutation for plasma lipid transport.

Molecular heterogeneity of classical and Duarte galactosemia: mutation analysis by denaturing gradient gel electrophoresis.

Classical galactosemia is caused by one common missense mutation (Q188R) and by several rare mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. The most common variant of GALT, the Duarte variant, occurs as two types, Duarte-1 (D-1) and Duarte-2 (D-2), both of which carry the sequence change N314D. D-1 increases, whereas D-2 decreases GALT activity. To study the molecular genetics of classical and Duarte galactosemia, we analyzed the GALT mutations in 30 families with classical galactosemia, in 10 families with the D-2 variant and in 3 individuals carrying the D-1 allele by denaturing gradient gel electrophoresis (DGGE). DGGE detected 59 of the 60 classical galactosemia alleles. Q188R accounted for 60%, K285N accounted for 28% of these alleles. Eight novel candidate galactosemia mutations were found. On all D-2 alleles N314D occurred in cis with two intronic sequence changes, on the D-1 alleles in cis with a neutral mutation in exon 7. We conclude that the mutations causing galactosemia are highly heterogeneous and that K285N is a second common galactosemia mutation in our population.

Evaluation of renal function in 60 long-term survivors of bone marrow transplantation.

Although the acute nephrotoxicity of bone marrow transplantation is well documented, long-term follow-up studies are scanty. Renal function was evaluated in 60 long-term survivors of allogeneic (24 patients) or autologous (36 patients) bone marrow transplantation (BMT) with a mean follow-up of 2 yr. Renal function tests included serum creatinine, GFR (inulin clearance), effective RPF (p-aminohyppurate clearance), urinary beta 2-microglobulin and N-acetyl-beta-D-glucosaminidase excretion, and renal tomography. The mean serum creatinine level was 83 +/- 3 and 93 +/- 3 mumol/L before and after grafting, respectively (P < 0.05). The mean GFR (93 +/- 3 mL/min) and effective RPF (419 +/- 16 mL/min) were significantly lower than in healthy controls (120 +/- 3 and 500 +/- 1 mL/min; P < 0.05) and than in candidates for BMT matched for age and hematologic diseases (117 +/- 3 and 469 +/- 11 mL/min; P < 0.05). Thirty-four patients had a fall in the GFR of at least 20%. Patients who received total body irradiation had significant lower creatinine clearance (86 +/- 3 mL/min) and GFR (86 +/- 3 mL/min) than the controls and the patients who received high-dose chemotherapy (100 +/- 4 and 104 +/- 5 mL/min; P < 0.05). These results suggest that subclinical renal dysfunction occurs frequently in marrow graft recipients, especially those who undergo total body irradiation.

Binding of gamma-aminobutyric acidA receptors to tubulin.

Tubulin cofractionated with gamma-aminobutyric acidA (GABAA) receptors upon affinity chromatography on Affigel 15 coupled to the benzodiazepine Ro 7-1986, and this cofractionation was not due to unspecific adsorption of tubulin to the column material. In addition, GABAA receptors not only bound to microtubules but also coassembled with added tubulin through three cycles of microtubule polymerization and depolymerization. These data indicate that GABAA receptors may be associated with the microtubule cytoskeleton in the brain. In an experiment designed to detect a protein that possibly could form a bridge between tubulin and the GABAA receptor, only a single protein band containing tubulin could be identified that was able to bind polymerized tubulin after sodium dodecyl sulfate-gel electrophoresis of affinity-purified GABAA receptors. These results are discussed with respect to a possible mechanism of association between GABAA receptors and microtubules.

Evidence for the existence of differential O-glycosylated alpha 5-subunits of the gamma-aminobutyric acidA receptor in the rat brain.

Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the gamma-aminobutyric acidA (GABAA) receptor alpha 5-subunit. These anti-peptide alpha 5(2-10) or anti-peptide alpha 5(427-433) antibodies reacted specifically with GABAA receptors purified from the brains of 5-10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N-Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O-Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide alpha 5(2-10) and the anti-peptide alpha 5(427-433) antibody. These results indicate the existence of at least two different alpha 5-subunits of the GABAA receptor that differ in their carbohydrate content. In contrast to other alpha- or beta-subunits of GABAA receptors so far investigated, at least one of these two alpha 5-subunits contains O-linked carbohydrates.