ABSTRACT
d-Glyceric aciduria (DGA) due to d-glycerate kinase deficiency (DGKD) is a rare autosomal-recessive inborn error of metabolism that is usually linked to the metabolism of fructose and serine. We describe a Moroccan patient with DGKD whose metabolic defect has been characterized by metabolite studies, sequencing of genomic DNA and by studies on the RNA level. Since birth the index patient presented with severe muscular hypotonia, joint hypermobility and tremor. Enantioselective analysis showed elevated d-glyceric acid in the urine of the patient, but not in that of his parents. DNA analysis revealed homozygosity in the GLYCTK gene for c.517G>T [p.(Val173Leu)], the first mutation reported for exon 3 of this gene, as well as for the c.530-4A>G polymorphism. RNA studies suggest that none of these sequence variants affects splicing. The mother was heterozygous for both sequence variants, the father heterozygous for the first one and homozygous for the polymorphism, which further supports that c.517G>T is the functionally relevant nucleotide change. The conservation of GLYCTK throughout evolution suggests an important biological role of this enzyme, although it is not known yet how mutations are linked to clinical features. Future studies should investigate the molecular defect in a more general way and search for additional roles of GLYCTK beyond its established role in catabolism of serine and fructose.
Subject(s)
Carbohydrate Metabolism, Inborn Errors/diagnosis , Carbohydrate Metabolism, Inborn Errors/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases/deficiency , Child , Exons/genetics , Glyceric Acids , Homozygote , Humans , Male , Metabolic Diseases/genetics , Mutation , Phosphotransferases/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA Splicing/genetics , Serine/geneticsABSTRACT
BACKGROUND: Valproic acid and its metabolites - particularly valproyl-CoA - are inhibitors of the enzyme N-acetylglutamate synthetase. The amino acid l-arginine can stimulate N-acetylglutamate synthetase activity and could be potentially used therapeutically to correct hyperammonemia caused by valproate therapy or overdose. Severely valproic-acid-poisoned patients are usually treated with l-carnitine or hemodialysis in order to decrease hyperammonemia. We herein report of five cases, in which l-arginine was administered. METHODS: Observational study on five cases. Patients with hyperammonemia (i.e., ammonia 80 > µg/dL) and symptoms consistent with valproate overdose (i.e., drowsiness, coma) were selected for treatment with l-arginine. Data was collected retrospectively. RESULTS: l-Arginine decreased ammonia levels in a close temporal relation (case I ammonia in EDTA-plasma [µg/dL] decreased from 381 to 39; case II from 281 to 50; case III from 669 to 74; case IV from 447 to 56; case V from 202 to 60). In cases I and II, hemodialysis was performed and l-carnitine was given before the administration of l-arginine. In case III, hemodialysis was performed after the administration of l-arginine was already started. In cases IV and V, treatment with l-arginine was the sole measure to decrease ammonia levels in plasma. CONCLUSION: The results suggest that l-arginine may be beneficial in selected cases of valproate overdose complicated by hyperammonemia. l-Arginine could extend our conventional treatment options for valproic acid overdose.
Subject(s)
Arginine/therapeutic use , Drug Overdose/drug therapy , Valproic Acid/poisoning , Acyl Coenzyme A/blood , Acyl Coenzyme A/poisoning , Adult , Amino-Acid N-Acetyltransferase/antagonists & inhibitors , Amino-Acid N-Acetyltransferase/blood , Ammonia/blood , Carnitine/therapeutic use , Coma/chemically induced , Coma/drug therapy , Drug Overdose/blood , Female , Humans , Hyperammonemia/blood , Hyperammonemia/drug therapy , Male , Renal Dialysis , Valproic Acid/bloodABSTRACT
d-Glyceric aciduria is caused by deficiency in d-glycerate kinase (GK) due to recessive mutations in the GLYCTK gene. GK catalyzes the conversion of d-glycerate to 2-phosphoglycerate which is an intermediary reaction in the catabolism of serine and fructose. Deficiency of GK leads to accumulation of d-glycerate, which may be detected in urine organic acid analysis. Debate exists as to whether this is a benign or disease-causing disorder as the reported phenotypes vary significantly. We report two siblings from a consanguineous Pakistani family. The index case is a 5year old boy with severe autism and global developmental delay. His urine organic acid analysis showed markedly increased excretion of glycerate, determined as d-form by enantioselective gas chromatography. There was no oxalic aciduria. His younger sister (3years old) is asymptomatic and developmentally normal (already bilingual). Her urine showed similar amounts of d-glycerate. Both children are homozygous for the novel mutation c.767C>G in exon 5 of the GLYCTK gene, predicted to affect the enzyme by replacing the evolutionarily conserved Proline with Arginine (P256R). Both parents are heterozygous carriers. These cases support the view that d-glycerate kinase deficiency is a benign disorder. Long term follow-up studies with a greater number of individuals may be required for further confirmation.
Subject(s)
Mutation/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Carbohydrate Metabolism, Inborn Errors/diagnostic imaging , Carbohydrate Metabolism, Inborn Errors/genetics , Child, Preschool , Family Health , Female , Humans , Magnetic Resonance Imaging , Male , Phosphotransferases/deficiency , Phosphotransferases/geneticsABSTRACT
Abstract 3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HMGCL, HMGCL) deficiency is a rare inborn error of ketogenesis. Even if the ketogenic enzyme is fully disrupted, an elevated signal for the ketone body acetoacetic acid is a frequent observation in the analysis of urinary organic acids, at least if derivatization is performed by methylation. We provide an explanation for this phenomenon and trace it back to degradation of the derivatized 3-hydroxy-3-methylglutaric acid and high temperature of the injector of the gas chromatograph.
ABSTRACT
BACKGROUND: Glutaric aciduria type 1 (GA1) is an inborn error in the metabolism of the amino acids tryptophan, lysine and hydroxylysine due to mutations in the GCDH gene coding for glutaryl-CoA dehydrogenase. Affected individuals often suffer from an encephalopathic crisis in infancy or childhood which results in acute striatal injury leading to a severe dystonic-dyskinetic movement disorder. Isobutyryl-coenzyme dehydrogenase (IBD) is an enzyme encoded by the ACAD8 gene and involved in the catabolism of the branched-chain amino acid valine. Both GA1 and IBD deficiency can be detected by expanded newborn screening using tandem-mass spectrometry, if they are considered screening targets. METHODS: Tandem-mass spectrometry and gas-chromatography with mass-selective detection were used for the assessment of key metabolites in body fluids of a patient with abnormal findings in newborn screening. Mutations were investigated by direct sequencing and by restriction fragment lengths analysis. Valine metabolism was studied in vitro in immortalized lymphocytes. RESULTS: Following accumulation of acylcarnitines C5DC and C4, of 3-hydroxyglutaric acid and isobutyrylglycine in body fluids, sequence analysis in the GCDH gene revealed homozygosity for a missense mutation in exon 6, c.482G>A, p.Arg161Gln, which had been reported in GA1 before. In the ACAD8 gene a novel mutation c.841+3G>C was identified, which results in loss of exon 7 and predicts a premature stop of translation. Impaired valine degradation was corroborated by the increased post-load level of acylcarnitine C4 in lymphocytes. CONCLUSION: The molecular basis of two inborn errors of metabolism in a newborn was elucidated. The metabolite studies underline the use of urinary C4 acylcarnitine as a sensitive marker of IBD deficiency. A functional test of IBD activity in lymphocytes may replace more invasive fibroblast studies. In view of the combination of two organic acidurias, which may both affect the level of free carnitine, careful follow-up including regular assessment of the carnitine status of the patient appears prudent.
Subject(s)
Glycine/analogs & derivatives , Amino Acid Metabolism, Inborn Errors/complications , Amino Acid Metabolism, Inborn Errors/diagnosis , Amino Acid Metabolism, Inborn Errors/genetics , Base Sequence , Brain Diseases, Metabolic/complications , Brain Diseases, Metabolic/diagnosis , Brain Diseases, Metabolic/genetics , DNA Mutational Analysis , Female , Follow-Up Studies , Glutaryl-CoA Dehydrogenase/deficiency , Glutaryl-CoA Dehydrogenase/genetics , Glycine/metabolism , Humans , Infant, Newborn , Male , Molecular Diagnostic TechniquesABSTRACT
BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein; CEACAM1) is expressed in normal bladder urothelium and in angiogenically activated endothelial cells, where it exhibits proangiogenic properties. OBJECTIVE: The aim of this study was to evaluate the value of urinary CEACAM1 for detection of urothelial carcinoma of the bladder (UCB). DESIGN, SETTING, AND PARTICIPANTS: This prospective study included 175 patients. MEASUREMENTS: Immunohistochemistry for CEACAM1 was performed on UCB sections of 10 patients. Enzyme-linked immunosorbent assay (ELISA) for CEACAM1 was performed on urine specimens of healthy volunteers (n=30), patients with benign prostatic hyperplasia (BPH; n=5), severe cystitis (n=5), non-muscle-invasive UCB (n=72), muscle-invasive UCB (n=21), or past history of UCB without evidence of disease (n=42). Western blot analysis was performed on a subgroup of these subjects (n=53). RESULTS AND LIMITATIONS: CEACAM1 immunostaining in normal urothelium disappears in noninvasive UCB but appears in endothelial cells of adjacent vessels. Western blotting revealed presence of CEACAM1 in the urine of no healthy volunteers, of 76% of noninvasive UCB patients, and of 100% of invasive UCB patients. ELISA analysis confirmed that urinary CEACAM1 levels were significantly higher in UCB patients compared with control subjects (median: 207 ng/ml vs 0 ng/ml; p<0.001). The area under the curve for UCB detection was 0.870 (95% confidence interval [CI]: 0.810-0.931). In multivariable logistic regression analyses that adjusted for the effects of age and gender, higher CEACAM1 levels were associated with cancer presence (hazard ratio [HR]: 2.89; 95% CI: 2.01-4.15; p<0.001) and muscle-invasive cancer (HR: 5.53; 95% CI: 1.68-18.24; p=0.005). The cut-off level of 110 ng/ml yielded sensitivity of 74% and specificity of 95% for detecting UCB. Sensitivity was 88% for detecting high-grade UCB and 100% for detecting invasive-stage UCB. Larger studies are necessary to establish the diagnostic and prognostic roles of this highly promising novel marker in UCB. CONCLUSIONS: Urinary CEACAM1 levels discriminate UCB patients from non-UCB subjects. Moreover, urinary levels of CEACAM1 increased with advancing stage and grade.
Subject(s)
Antigens, CD/urine , Biomarkers, Tumor/urine , Carcinoma/diagnosis , Cell Adhesion Molecules/urine , Urinary Bladder Neoplasms/diagnosis , Urinary Bladder/chemistry , Blotting, Western , Carcinoma/pathology , Carcinoma/urine , Case-Control Studies , Chi-Square Distribution , Endothelial Cells/chemistry , Enzyme-Linked Immunosorbent Assay , Germany , Humans , Immunohistochemistry , Logistic Models , Neoplasm Invasiveness , Neoplasm Staging , Odds Ratio , Predictive Value of Tests , Prognosis , Prospective Studies , Risk Assessment , Risk Factors , Sensitivity and Specificity , Up-Regulation , Urinary Bladder/pathology , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/urine , Urothelium/chemistryABSTRACT
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelia, blood vessel endothelia, and leukocytes. A variety of physiological functions have been assigned to CEACAM1. It is involved in the formation of glands and blood vessels, in immune reactions, and in the regulation of tumor growth. As a homophilic and heterophilic adhesion receptor, it signals through different cellular pathways. The existence of special oligosaccharide structures such as Lewis x or sialyl-Lewis x glycans within this highly glycosylated protein has been postulated, but chemical proof is missing so far. Because such structures are known to be essential for different cell-cell recognition and adhesion processes, characterizing the CEACAM1 glycan structure is of pivotal importance in revealing the biological function of CEACAM1. We examine the terminal glycosylation pattern of CEACAM1 from human granulocytes, focusing on Lewis x epitopes. Lewis x-specific antibodies react with immunoaffinity-purified native CEACAM1. Antibody binding was completely abolished by treatment with fucosidase III, confirming a terminal alpha(1-3,4) fucose linkage to the N-acetylglucosamine of lactosamine residues, a key feature of Lewis epitopes. To verify these data, MALDI-TOF MS analysis after stepwise exoglycosidase digestion of the CEACAM1 N-glycan mixture was performed. A complex mixture of CEACAM1-bound oligosaccharides could be characterized with an unusually high amount of fucose. The sequential digestions clearly identified several different Lewis x glycan epitopes, which may modulate the cell adhesive functions of CEACAM1.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation/metabolism , Granulocytes/metabolism , Oligosaccharides/metabolism , Antibodies/immunology , Antigens, CD/chemistry , Antigens, CD/isolation & purification , Antigens, Differentiation/chemistry , Antigens, Differentiation/isolation & purification , Blotting, Western , Cell Adhesion Molecules , Cell Line , Glycoside Hydrolases/metabolism , Humans , Monosaccharides/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Sialyl Lewis X Antigen , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Here, we demonstrate the expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in angiogenic sprouts but not in large mother blood vessels within tumor tissue. Correspondingly, only human microvascular endothelial cells involved in in vitro tube formation exhibit CEACAM1. CEACAM1-overexpressing versus CEACAM1-silenced human microvascular endothelial cells were used in migration and tube formation assays. CEACAM1-overexpressing microvascular endothelial cells showed prolonged survival and increased tube formation when they were stimulated with vascular endothelial growth factor (VEGF), whereas CEACAM1 silencing via small interfering RNA blocks these effects. Gene array and LightCycler analyses show an up-regulation of angiogenic factors such as VEGF, VEGF receptor 2, angiopoietin-1, angiopoietin-2, tie-2, angiogenin, and interleukin-8 but a down-regulation of collagen XVIII/endostatin and Tie-1 in CEACAM1-overexpressing microvascular endothelial cells. Western blot analyses confirm these results for VEGF and endostatin at the protein level. These results suggest that constitutive expression of CEACAM1 in microvascular endothelial cells switches them to an angiogenic phenotype, whereas CEACAM1 silencing apparently abrogates the VEGF-induced morphogenetic effects during capillary formation. Thus, strategies targeting the endothelial up-regulation of CEACAM1 might be promising for antiangiogenic tumor therapy.
Subject(s)
Antigens, CD/physiology , Antigens, Differentiation/physiology , Endothelium, Vascular/physiology , Neovascularization, Physiologic/physiology , Signal Transduction/physiology , Base Sequence , Cell Adhesion Molecules , Cell Survival/physiology , Cells, Cultured , Collagen Type XVIII/physiology , DNA Primers , Down-Regulation/physiology , Endostatins/physiology , Endothelium, Vascular/cytology , Humans , Receptor, TIE-1/physiologyABSTRACT
Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is usually expressed at the luminal surface of different epithelia and is up-regulated in endothelial cells during angiogenesis. Here, we demonstrate evidence of morphogenetic effects of CEACAM1 in spermatogenesis. CEACAM1 is detectable in normal testicular tissue and seminal fluid. It is present in the adluminal part of Sertoli cells extending only as far as the tight junctions between them. CEACAM1 immunostaining is significantly increased and extends to the basal part of Sertoli cells in the presence of carcinoma in situ. Also, in vitro-induced spermatogenetic disturbance leads to an enhanced CEACAM1 expression in Sertoli cells after 3 days of culture. Remarkably, seminiferous tubules containing exclusively Sertoli cells do not exhibit any CEACAM1 expression. CEACAM1 staining was absent in vascular endothelial cells of normal testicular tissue, but present in small blood vessels of seminomas. These data suggest that CEACAM1 expression in Sertoli cells depends on the presence of germ cells and plays a role in adhesive interactions between Sertoli and differentiating germ cells. Its up-regulation in Sertoli cells accompanying spermatogenic damage may contribute to reconstruction and maintenance of the tubular structure of seminiferous tubules. Additionally, CEACAM1 is apparently involved in the angiogenesis of germ cell tumours.