40 results on '"Rüfenacht, V"'
Search Results
2. Psychosocial development during school period of children operated for transposition of the great arteries
- Author
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Stucki, E., primary, Stocker, F. P., additional, Hämmerli, H., additional, Rüfenacht, V., additional, Stucki, M., additional, Weber, J. W., additional, and Schüpbach, P., additional
- Published
- 1992
- Full Text
- View/download PDF
3. N-Acetylglutamate Synthase Deficiency Due to a Recurrent Sequence Variant in the N-acetylglutamate Synthase Enhancer Region
- Author
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Williams, M. (Monique), Burlina, A.B. (Alberto), Rubert, L. (Laura), Polo, G. (Giulia), Ruijter, G.J.G. (George), Born, M. (Myrthe) van den, Rüfenacht, V. (Véronique), Haskins, N. (Nantaporn), Zutven, L.J.C.M. (Laura) van, Tuchman, M. (Mendel), Saris, J.J. (Jasper J.), Häberle, J. (Johannes), Caldovic, L. (Ljubica), Williams, M. (Monique), Burlina, A.B. (Alberto), Rubert, L. (Laura), Polo, G. (Giulia), Ruijter, G.J.G. (George), Born, M. (Myrthe) van den, Rüfenacht, V. (Véronique), Haskins, N. (Nantaporn), Zutven, L.J.C.M. (Laura) van, Tuchman, M. (Mendel), Saris, J.J. (Jasper J.), Häberle, J. (Johannes), and Caldovic, L. (Ljubica)
- Abstract
N-acetylglutamate synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C > T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C > T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C > T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C > T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD.
- Published
- 2018
- Full Text
- View/download PDF
4. Increased expression of the proton-sensing G protein-coupled receptor Gpr65 during retinal degeneration
- Author
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Ail, D., primary, Rüfenacht, V., additional, Caprara, C., additional, Samardzija, M., additional, Kast, B., additional, and Grimm, C., additional
- Published
- 2015
- Full Text
- View/download PDF
5. Mutation analysis of urea cycle disorders.
- Author
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Rüfenacht, V\'eronique and H\"aberle, Johannes
- Subjects
- *
GENETIC mutation measurement , *METABOLIC disorders , *HYPERAMMONEMIA , *GENETIC counseling , *PRENATAL diagnosis - Abstract
Urea cycle disorders (UCDs) are a group of autosomal or X-linked, recessively inherited errors of metabolism that lead to severe neurological disease due to insufficient detoxification of excess nitrogen. The resulting hyperammonemia is the key feature of UCDs, but at the same time only a surrogate marker. In the majority of cases, a sole biochemical analysis is indicative but not diagnostic. Therefore, additional means are required and mutation analysis is the method of choice for confirmation in most cases of UCD. In addition to confirming the diagnosis, mutation analysis enables genetic counseling and prenatal testing and contributes to new research approaches. All genes involved in any of the enzymatic (namely the genes for N-acetylglutamate synthase, NAGS; carbamoylphosphate synthetase, CPS1; ornithine transcarbamylase, OTC; argininosuccinate synthetase, ASS1; argininosuccinate lyase, ASL; arginase, ARG1) or transporter steps (namely the genes for the ornithine/citrulline antiporter ORNT1, SLC25A15; glutamate/aspartate antiporter citrin, SLC25A13) of the urea cycle are known and thus accessible for genetic testing. In most situations, direct Sanger sequencing using DNA from peripheral blood cells can be done but there are exceptions to this such as in the case of the relatively large CPS1 gene which renders RNA based mutation analysis an attractive alternative. With this review, we provide an overview on the current methods used for mutation analysis of each UCD gene, we mention possible pitfalls and their solutions, and discuss alternative methods which may become standard in the future. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
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6. The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery
- Author
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Véronique Rüfenacht, Johannes A. Mayr, Michele Costanzo, Johannes Häberle, Søren W Gersting, Margherita Ruoppolo, Noémie Remacle, Clothilde Roux, Martin Poms, Madalena Barroso, Marianna Caterino, René G. Feichtinger, Hong-Phuc Cudré-Cung, Mary Gonzalez Melo, Olivier Braissant, Cristina Cudalbu, Diana Ballhausen, Gonzalez Melo, M, Remacle, N, Cudré-Cung, Hp, Roux, C, Poms, M, Cudalbu, C, Barroso, M, Gersting, Sw, Feichtinger, Rg, Mayr, Ja, Costanzo, M, Caterino, M, Ruoppolo, M, Rüfenacht, V, Häberle, J, Braissant, O, Ballhausen, D., University of Zurich, and Ballhausen, Diana
- Subjects
0301 basic medicine ,1303 Biochemistry ,Arginine ,Endocrinology, Diabetes and Metabolism ,030105 genetics & heredity ,Biochemistry ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,organic-acids ,Hyperammonemia ,Microglial activation ,Gene Knock-In Techniques ,Gliosis ,Glutaryl-CoA Dehydrogenase ,Chemistry ,Brain Diseases, Metabolic ,Brain ,food-intake ,Pathophysiology ,1310 Endocrinology ,Diabetes and Metabolism ,2712 Endocrinology, Diabetes and Metabolism ,Urea cycle ,astrogliosis ,medicine.medical_specialty ,mice ,Normal diet ,mouse model ,610 Medicine & health ,Creatine ,energy-metabolism ,Astrogliosi ,pipecolic acid ,03 medical and health sciences ,1311 Genetics ,Internal medicine ,1312 Molecular Biology ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Amino Acid Metabolism, Inborn Errors ,Lysine ,lysine metabolism ,Glutaric aciduria ,natural-history ,Glutaric aciduria type I ,mutations ,medicine.disease ,Rats ,Cerebral organic aciduria ,Lysine degradation ,Disease Models, Animal ,10036 Medical Clinic ,Inborn error of metabolism ,030217 neurology & neurosurgery ,Metabolism, Inborn Errors - Abstract
Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra-and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new ther-apeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery., (c) 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
- Published
- 2021
7. Expression and function of the urea cycle in widely-used hepatic cellular models.
- Author
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Makris G, Veit L, Rüfenacht V, Klassa S, Zürcher N, Matsumoto S, Poms M, and Häberle J
- Subjects
- Humans, Induced Pluripotent Stem Cells metabolism, Liver metabolism, Cell Differentiation, Arginase metabolism, Hep G2 Cells, Urea metabolism, Urea Cycle Disorders, Inborn metabolism, Hepatocytes metabolism
- Abstract
The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma-derived cell lines, induced pluripotent stem cell hepatocytes (iPSC-Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma-derived, 2-week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC-Heps, which are known to exhibit batch-to-batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs., (© 2024 SSIEM.)
- Published
- 2024
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8. Impact of small molecule-mediated inhibition of ammonia detoxification on lung malignancies and liver metabolism.
- Author
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Makris G, Kayhan S, Kreuzer M, Rüfenacht V, Faccin E, Underhaug J, Diez-Fernandez C, Knobel PA, Poms M, Gougeard N, Rubio V, Martinez A, Pruschy M, and Häberle J
- Subjects
- Humans, Liver, Ammonia metabolism, Lung Neoplasms metabolism
- Published
- 2023
- Full Text
- View/download PDF
9. O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis.
- Author
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Soria LR, Makris G, D'Alessio AM, De Angelis A, Boffa I, Pravata VM, Rüfenacht V, Attanasio S, Nusco E, Arena P, Ferenbach AT, Paris D, Cuomo P, Motta A, Nitzahn M, Lipshutz GS, Martínez-Pizarro A, Richard E, Desviat LR, Häberle J, van Aalten DMF, and Brunetti-Pierri N
- Subjects
- Acetylglucosamine, Animals, Biocatalysis, Disease Models, Animal, Glycosylation, Humans, Mammals metabolism, Mice, N-Acetylglucosaminyltransferases genetics, N-Acetylglucosaminyltransferases metabolism, Propionic Acidemia genetics, Propionic Acidemia metabolism, Protein Processing, Post-Translational genetics, Ammonia metabolism, Carbamoyl-Phosphate Synthase (Ammonia) genetics, Carbamoyl-Phosphate Synthase (Ammonia) metabolism, Hyperammonemia genetics, Hyperammonemia metabolism, Urea metabolism, Uridine Diphosphate genetics, Uridine Diphosphate metabolism
- Abstract
Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases., (© 2022. The Author(s).)
- Published
- 2022
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10. Aquaporin 9 induction in human iPSC-derived hepatocytes facilitates modeling of ornithine transcarbamylase deficiency.
- Author
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Laemmle A, Poms M, Hsu B, Borsuk M, Rüfenacht V, Robinson J, Sadowski MC, Nuoffer JM, Häberle J, and Willenbring H
- Subjects
- Adult, Hepatocytes metabolism, Humans, Urea, Aquaporins metabolism, Induced Pluripotent Stem Cells metabolism, Liver Diseases metabolism, Ornithine Carbamoyltransferase Deficiency Disease genetics, Ornithine Carbamoyltransferase Deficiency Disease metabolism, Ornithine Carbamoyltransferase Deficiency Disease therapy
- Abstract
Background and Aims: Patient-derived human-induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes (hiPSC-Heps) have facilitated the study of rare genetic liver diseases. Here, we aimed to establish an in vitro liver disease model of the urea cycle disorder ornithine transcarbamylase deficiency (OTCD) using patient-derived hiPSC-Heps., Approach and Results: Before modeling OTCD, we addressed the question of why hiPSC-Heps generally secrete less urea than adult primary human hepatocytes (PHHs). Because hiPSC-Heps are not completely differentiated and maintain some characteristics of fetal PHHs, we compared gene-expression levels in human fetal and adult liver tissue to identify genes responsible for reduced urea secretion in hiPSC-Heps. We found lack of aquaporin 9 (AQP9) expression in fetal liver tissue as well as in hiPSC-Heps, and showed that forced expression of AQP9 in hiPSC-Heps restores urea secretion and normalizes the response to ammonia challenge by increasing ureagenesis. Furthermore, we proved functional ureagenesis by challenging AQP9-expressing hiPSC-Heps with ammonium chloride labeled with the stable isotope [
15 N] (15 NH4 Cl) and by assessing enrichment of [15 N]-labeled urea. Finally, using hiPSC-Heps derived from patients with OTCD, we generated a liver disease model that recapitulates the hepatic manifestation of the human disease. Restoring OTC expression-together with AQP9-was effective in fully correcting OTC activity and normalizing ureagenesis as assessed by15 NH4 Cl stable-isotope challenge., Conclusion: Our results identify a critical role for AQP9 in functional urea metabolism and establish the feasibility of in vitro modeling of OTCD with hiPSC-Heps. By facilitating studies of OTCD genotype/phenotype correlation and drug screens, our model has potential for improving the therapy of OTCD., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)- Published
- 2022
- Full Text
- View/download PDF
11. Recovery of enzyme activity in biotinidase deficient individuals during early childhood.
- Author
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Forny P, Wicht A, Rüfenacht V, Cremonesi A, and Häberle J
- Subjects
- Biotin therapeutic use, Biotinidase genetics, Biotinidase metabolism, Child, Preschool, Genetic Testing, Humans, Infant, Newborn, Mutation, Neonatal Screening, Biotinidase Deficiency diagnosis, Biotinidase Deficiency drug therapy, Biotinidase Deficiency genetics
- Abstract
Deficiency of the biotinidase (BTD) enzyme is an inborn error of biotin metabolism caused by biallelic pathogenic variants in the BTD gene. There are two forms, partial and profound BTD deficiency, which both can be successfully treated with pharmacological doses of biotin, justifying the inclusion of this disorder in the newborn screening in numerous countries. We investigated the BTD deficiency cohort (N = 87) in our metabolic center, as it was detected upon newborn screening since 2005, and aimed to better understand the long-term course of BTD enzyme activity and how it may relate to the patients' genetic background. We observed that individuals with partial BTD deficiency display an elevation of BTD enzyme activity with increasing age in 48% of cases-a recovery which allowed adjustment or stop of biotin supplementation in 20% of all individuals. In addition, we were able to recruit 56 patients (64%) for genetic testing, revealing 19 different variants (2 novel), and constituting 22 different genotypes. Genotype-phenotype correlations revealed that the most abundant allele in our cohort p.(Asp444His) was also the most common variant in patients displaying recovery of BTD enzyme activity. Based on our results, we recommend to retest all patients with partial BTD deficiency at the age of 5 years, as this may result in an impact on therapy. Moreover, genetic testing of BTD deficient individuals can allow prediction of the severity of BTD deficiency and of the likelihood of BTD enzyme activity recovery with age., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)
- Published
- 2022
- Full Text
- View/download PDF
12. Noncoding sequence variants define a novel regulatory element in the first intron of the N-acetylglutamate synthase gene.
- Author
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Häberle J, Moore MB, Haskins N, Rüfenacht V, Rokicki D, Rubio-Gozalbo E, Tuchman M, Longo N, Yandell M, Andrews A, AhMew N, and Caldovic L
- Subjects
- Humans, Introns, Regulatory Sequences, Nucleic Acid, Amino-Acid N-Acetyltransferase chemistry, Amino-Acid N-Acetyltransferase genetics, Hyperammonemia genetics, Urea Cycle Disorders, Inborn genetics
- Abstract
N-acetylglutamate synthase deficiency is an autosomal recessive urea cycle disorder caused either by decreased expression of the NAGS gene or defective NAGS enzyme resulting in decreased production of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1). NAGSD is the only urea cycle disorder that can be effectively treated with a single drug, N-carbamylglutamate (NCG), a stable NAG analog, which activates CPS1 to restore ureagenesis. We describe three patients with NAGSD due to four novel noncoding sequence variants in the NAGS regulatory regions. All three patients had hyperammonemia that resolved upon treatment with NCG. Sequence variants NM_153006.2:c.427-222G>A and NM_153006.2:c.427-218A>C reside in the 547 bp-long first intron of NAGS and define a novel NAGS regulatory element that binds retinoic X receptor α. Sequence variants NC_000017.10:g.42078967A>T (NM_153006.2:c.-3065A>T) and NC_000017.10:g.42078934C>T (NM_153006.2:c.-3098C>T) reside in the NAGS enhancer, within known HNF1 and predicted glucocorticoid receptor binding sites, respectively. Reporter gene assays in HepG2 and HuH-7 cells demonstrated that all four substitutions could result in reduced expression of NAGS. These findings show that analyzing noncoding regions of NAGS and other urea cycle genes can reveal molecular causes of disease and identify novel regulators of ureagenesis., (© 2021 Wiley Periodicals LLC.)
- Published
- 2021
- Full Text
- View/download PDF
13. The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.
- Author
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Gonzalez Melo M, Remacle N, Cudré-Cung HP, Roux C, Poms M, Cudalbu C, Barroso M, Gersting SW, Feichtinger RG, Mayr JA, Costanzo M, Caterino M, Ruoppolo M, Rüfenacht V, Häberle J, Braissant O, and Ballhausen D
- Subjects
- Amino Acid Metabolism, Inborn Errors metabolism, Amino Acid Metabolism, Inborn Errors pathology, Animals, Arginine metabolism, Brain pathology, Brain Diseases, Metabolic metabolism, Brain Diseases, Metabolic pathology, Creatine blood, Disease Models, Animal, Gene Knock-In Techniques, Gliosis metabolism, Gliosis pathology, Glutaryl-CoA Dehydrogenase metabolism, Humans, Lysine metabolism, Metabolism, Inborn Errors genetics, Metabolism, Inborn Errors metabolism, Rats, Amino Acid Metabolism, Inborn Errors genetics, Brain metabolism, Brain Diseases, Metabolic genetics, Gliosis genetics, Glutaryl-CoA Dehydrogenase deficiency, Glutaryl-CoA Dehydrogenase genetics
- Abstract
Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdh
ki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
14. Maple syrup urine disease: Clinical outcomes, metabolic control, and genotypes in a screened population after four decades of newborn bloodspot screening in the Republic of Ireland.
- Author
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O'Reilly D, Crushell E, Hughes J, Ryan S, Rogers Y, Borovickova I, Mayne P, Riordan M, Awan A, Carson K, Hunter K, Lynch B, Shahwan A, Rüfenacht V, Häberle J, Treacy EP, Monavari AA, and Knerr I
- Subjects
- Adolescent, Child, Child, Preschool, Diet, Protein-Restricted, Dried Blood Spot Testing, Early Diagnosis, Female, Genotype, Humans, Infant, Infant, Newborn, Ireland, Leucine blood, Male, Neonatal Screening methods, Phenotype, Retrospective Studies, Maple Syrup Urine Disease diagnosis, Maple Syrup Urine Disease genetics
- Abstract
Since 1972, 18 patients (10 females/8 males) have been detected by newborn bloodspot screening (NBS) with neonatal-onset maple syrup urine disease (MSUD) in Ireland. Patients were stratified into three clusters according to clinical outcome at the time of data collection, including developmental, clinical, and IQ data. A fourth cluster comprised of two early childhood deaths; a third patient died as an adult. We present neuroimaging and electroencephalography together with clinical and biochemical data. Incidence of MSUD (1972-2018) was 1 in 147 975. Overall good clinical outcomes were achieved with 15/18 patients alive and with essentially normal functioning (with only the lowest performing cluster lying beyond a single SD on their full scale intelligence quotient). Molecular genetic analysis revealed genotypes hitherto not reported, including a possible digenic inheritance state for the BCKDHA and DBT genes in one family. Treatment has been based on early implementation of emergency treatment, diet, close monitoring, and even dialysis in the setting of acute metabolic decompensation. A plasma leucine ≥400 μmol/L (outside therapeutic range) was more frequently observed in infancy or during adolescence, possibly due to infections, hormonal changes, or noncompliance. Children require careful management during metabolic decompensations in early childhood, and this represented a key risk period in our cohort. A high level of metabolic control can be achieved through diet with early implementation of a "sick day" regime and, in some cases, dialysis as a rescue therapy. The Irish cohort, despite largely classical phenotypes, achieved good outcomes in the NBS era, underlining the importance of early diagnosis and skilled multidisciplinary team management., (© 2020 SSIEM.)
- Published
- 2021
- Full Text
- View/download PDF
15. Clinical and structural insights into potential dominant negative triggers of proximal urea cycle disorders.
- Author
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Makris G, Lauber M, Rüfenacht V, Gemperle C, Diez-Fernandez C, Caldovic L, Froese DS, and Häberle J
- Subjects
- Amino Acid Substitution, Female, Heterozygote, Homozygote, Humans, Male, Protein Domains, Amino-Acid N-Acetyltransferase chemistry, Amino-Acid N-Acetyltransferase genetics, Amino-Acid N-Acetyltransferase metabolism, Carbamoyl-Phosphate Synthase (Ammonia) chemistry, Carbamoyl-Phosphate Synthase (Ammonia) genetics, Carbamoyl-Phosphate Synthase (Ammonia) metabolism, Genes, Dominant, Mutation, Missense, Ornithine Carbamoyltransferase chemistry, Ornithine Carbamoyltransferase genetics, Ornithine Carbamoyltransferase metabolism, Urea Cycle Disorders, Inborn enzymology, Urea Cycle Disorders, Inborn genetics
- Abstract
Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as "heterozygous-only" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
16. Response to Baertling et al.
- Author
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Häberle J and Rüfenacht V
- Subjects
- Bicarbonates, Carbonic Anhydrases
- Published
- 2020
- Full Text
- View/download PDF
17. Improvement of diagnostic yield in carbamoylphosphate synthetase 1 ( CPS1 ) molecular genetic investigation by RNA sequencing.
- Author
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Isler J, Rüfenacht V, Gemperle C, Allegri G, and Häberle J
- Abstract
Carbamoylphosphate synthetase 1 (CPS1) deficiency is a rare inborn error of metabolism leading often to neonatal onset hyperammonemia with coma and high mortality. The biochemical features of the disease are nonspecific and cannot distinguish this condition from other defects of the urea cycle, namely N -acetylglutamate synthase deficiency. Therefore, molecular genetic investigation is required for confirmation of the disease, and nowadays this is done with increasing frequency applying next-generation sequencing (NGS) techniques. Our laboratory has a long-standing interest in CPS1 molecular genetic investigation and receives samples from centers in Europe and many other countries. We perform RNA-based CPS1 molecular genetic investigation as first line investigation and wanted in this study to evaluate our experience with this approach as compared to NGS. In the past 15 years, 297 samples were analyzed, which were referred from 37 countries. CPS1 deficiency could be confirmed in 155 patients carrying 136 different genotypes with only a single mutation recurring more than two times. About 10% of the total 172 variants comprised complex changes (eg, intronic changes possibly affecting splicing, deletions, insertions, or deletions_insertions), which would have been partly missed if only NGS was done. Likewise, RNA analysis was crucial for correct interpretation of at least half of the complex mutations. This study gives highest sensitivity to RNA-based CPS1 molecular genetic investigation and underlines that NGS should be done together with copy number variation analysis. We propose that unclear cases should be investigated by RNA sequencing in addition, if this method is not used as the initial diagnostic procedure., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)
- Published
- 2020
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- View/download PDF
18. A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.
- Author
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Khoja S, Nitzahn M, Truong B, Lambert J, Willis B, Allegri G, Rüfenacht V, Häberle J, and Lipshutz GS
- Subjects
- Animals, Animals, Newborn, Carbamoyl-Phosphate Synthase (Ammonia) deficiency, Carbamoyl-Phosphate Synthase I Deficiency Disease blood, Carbamoyl-Phosphate Synthase I Deficiency Disease genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutation, Carbamoyl-Phosphate Synthase (Ammonia) genetics, Carbamoyl-Phosphate Synthase I Deficiency Disease complications, Carbamoyl-Phosphate Synthase I Deficiency Disease mortality, Glutamine blood, Hyperammonemia blood, Hyperammonemia complications, Hyperammonemia genetics, Hyperammonemia mortality
- Abstract
The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1
-/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder., (© 2019 SSIEM.)- Published
- 2019
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19. A liver-humanized mouse model of carbamoyl phosphate synthetase 1-deficiency.
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Srinivasan RC, Zabulica M, Hammarstedt C, Wu T, Gramignoli R, Kannisto K, Ellis E, Karadagi A, Fingerhut R, Allegri G, Rüfenacht V, Thöny B, Häberle J, Nuoffer JM, and Strom SC
- Subjects
- Animals, Carbamoyl-Phosphate Synthase (Ammonia) metabolism, Cells, Cultured, Child, Disease Models, Animal, Female, Hepatocytes metabolism, Humans, Hydrolases metabolism, Infant, Infant, Newborn, Liver pathology, Male, Mice, Mice, Transgenic, Middle Aged, Organ Specificity genetics, Carbamoyl-Phosphate Synthase (Ammonia) genetics, Carbamoyl-Phosphate Synthase I Deficiency Disease genetics, Carbamoyl-Phosphate Synthase I Deficiency Disease pathology, Hepatocytes transplantation, Hydrolases genetics, Liver metabolism
- Abstract
A liver-humanized mouse model for CPS1-deficiency was generated by the high-level repopulation of the mouse liver with CPS1-deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1-proficient human hepatocytes, mice that are repopulated with CPS1-deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [
15 N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease-affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1-deficiency. Liver-humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease-affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene-editing technologies directed to human hepatocytes and the translation of gene-editing technology to the clinic., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)- Published
- 2019
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20. N-Acetylglutamate Synthase Deficiency Due to a Recurrent Sequence Variant in the N-acetylglutamate Synthase Enhancer Region.
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Williams M, Burlina A, Rubert L, Polo G, Ruijter GJG, van den Born M, Rüfenacht V, Haskins N, van Zutven LJCM, Tuchman M, Saris JJ, Häberle J, and Caldovic L
- Subjects
- Amino-Acid N-Acetyltransferase metabolism, Base Sequence, Child, Child, Preschool, Female, Humans, Hyperammonemia, Prognosis, Urea Cycle Disorders, Inborn metabolism, Urea Cycle Disorders, Inborn pathology, Amino-Acid N-Acetyltransferase genetics, Enhancer Elements, Genetic, Genetic Variation, Urea Cycle Disorders, Inborn etiology
- Abstract
N-acetylglutamate synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C > T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C > T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C > T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C > T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD.
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- 2018
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21. Metabolic follow-up of a Croatian patient with gyrate atrophy and a new mutation in the OAT gene: a case report.
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Zekušić M, Škaričić A, Fumić K, Rogić D, Žigman T, Petković Ramadža D, Vukojević N, Rüfenacht V, Uroić V, and Barić I
- Subjects
- Blood Cell Count, Child, Croatia, Female, Fluorescein Angiography, Follow-Up Studies, Gyrate Atrophy blood, Gyrate Atrophy diagnostic imaging, Gyrate Atrophy enzymology, Humans, Tomography, Optical Coherence, Gyrate Atrophy genetics, Mutation, Ornithine-Oxo-Acid Transaminase genetics
- Abstract
Gyrate atrophy (GA) of the choroid and retina is a rare autosomal recessive disorder that occurs due to deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT). Hyperornithinemia causes degeneration of the retina with symptoms like myopia, reduced night vision and progressive vision loss. Our patient is a 10-year-old girl with impaired vision and strabismus. As part of the metabolic work-up, plasma amino acid analysis revealed significantly increased concentration of ornithine (1039 μmol/L; reference interval 20 - 155 μmol/L). Molecular genetic analysis revealed homozygous mutation in exon 7 of the OAT gene that has not been reported previously (c.868_870delCTT p.(Leu290del)). This in frame deletion was predicted to be deleterious by in silico software analysis. Our patient was treated with pyridoxine (vitamin B
6 in a dose of 2 x 100 mg/day), low-protein diet (0.6 g/kg/day) and L-lysine supplementation which resulted in a significant reduction in plasma ornithine concentrations to 53% of the initial concentration and the ophthalmologic findings showed significant improvement. We conclude that low protein diet and lysine supplementation can lead to long-term reduction in plasma ornithine concentrations and, if started at an early age, notably slow the progression of retinal function loss in patients with GA. The effect of therapy can be reliably monitored by periodical measurement of plasma ornithine concentration. To our knowledge, this is the first report of OAT deficiency in Croatia., Competing Interests: Potential conflict of interest: None declared.- Published
- 2018
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22. Urea cycle disorders in India: clinical course, biochemical and genetic investigations, and prenatal testing.
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Bijarnia-Mahay S, Häberle J, Jalan AB, Puri RD, Kohli S, Kudalkar K, Rüfenacht V, Gupta D, Maurya D, Verma J, Shigematsu Y, Yamaguchi S, Saxena R, and Verma IC
- Subjects
- Citrullinemia diagnosis, Citrullinemia genetics, Female, Humans, Hyperammonemia diagnosis, Hyperammonemia genetics, Male, Mutation genetics, Ornithine Carbamoyltransferase Deficiency Disease diagnosis, Ornithine Carbamoyltransferase Deficiency Disease genetics, Point Mutation genetics, Prenatal Diagnosis methods, Urea Cycle Disorders, Inborn genetics, Urea Cycle Disorders, Inborn diagnosis
- Abstract
Background: Urea cycle disorders (UCDs) are inherited metabolic disorders that present with hyperammonemia, and cause significant mortality and morbidity in infants and children. These disorders are not well reported in the Indian population, due to lack of a thorough study of the clinical and molecular profile., Results: We present data from two major metabolic centres in India, including 123 cases of various UCDs. The majority of them (72/123, 58%) presented in the neonatal period (before 30 days of age) with 88% on or before day 7 of life (classical presentation), and had a high mortality (64/72, 88%). Citrullinemia type 1 was the most common UCD, observed in 61/123 patients. Ornithine transcarbamylase (OTC) deficiency was the next most common, seen in 24 cases. Argininosuccinic aciduria was diagnosed in 20 cases. Deficiencies of arginase, N-acetylglutamate synthase, carbamoyl phosphate synthetase, citrin, and lysinuric protein intolerance were also observed. Molecular genetic analysis revealed two common ASS1 mutations: c.470G > A (p.Arg157His) and c.1168G > A (p.Gly390Arg) (36 of 55 tested patients). In addition, few recurrent point mutations in ASL gene, and a deletion of the whole OTC gene were also noted. A total of 24 novel mutations were observed in the various genes studied. We observed a poor clinical outcome with an overall all time mortality of 63% (70/110 cases with a known follow-up), and disability in 70% (28/40) among the survivors. Prenatal diagnosis was performed in 30 pregnancies in 25 families, including one pre-implantation genetic diagnosis., Conclusions: We report the occurrence of UCDs in India and the spectrum that may be different from the rest of the world. Citrullinemia type 1 was the most common UCD observed in the cohort. Increasing awareness amongst clinicians will improve outcomes through early diagnosis and timely treatment. Genetic diagnosis in the proband will enable prenatal/pre-implantation diagnosis in subsequent pregnancies.
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- 2018
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23. Mutations and common variants in the human arginase 1 (ARG1) gene: Impact on patients, diagnostics, and protein structure considerations.
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Diez-Fernandez C, Rüfenacht V, Gemperle C, Fingerhut R, and Häberle J
- Subjects
- Brazil, China, Codon, Nonsense genetics, Humans, Mutation, Missense genetics, Turkey, Arginase genetics, Mutation genetics
- Abstract
The urea cycle disorder argininemia is caused by a defective arginase 1 (ARG1) enzyme resulting from mutations in the ARG1 gene. Patients generally develop hyperargininemia, spastic paraparesis, progressive neurological and intellectual impairment, and persistent growth retardation. Interestingly, in contrast to other urea cycle disorders, hyperammonemia is rare. We report here 66 mutations (12 of which are novel), including 30 missense mutations, seven nonsense, 10 splicing, 15 deletions, two duplications, one small insertion, and one translation initiation codon mutation. For the most common mutations (p.Thr134Ile, p.Gly235Arg and p.Arg21*), which cluster geographically in Brazil, China, or Turkey, a structural rationalization of their effect has been included. In order to gain more knowledge on the disease, we have collected clinical and biochemical information of 112 patients, including the patients' genetic background and ethnic origin. We have listed as well the missense variants with unknown relevance. For all missense variants (of both known and unknown relevance), the conservation, severity prediction, and ExAc scores have been included. Lastly, we review ARG1 regulation, animal models, diagnostic strategies, newborn screening, prenatal testing, and treatment options., (© 2018 Wiley Periodicals, Inc.)
- Published
- 2018
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24. Heterogeneous clinical spectrum of DNAJC12-deficient hyperphenylalaninemia: from attention deficit to severe dystonia and intellectual disability.
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van Spronsen FJ, Himmelreich N, Rüfenacht V, Shen N, Vliet DV, Al-Owain M, Ramzan K, Alkhalifi SM, Lunsing RJ, Heiner-Fokkema RM, Rassi A, Gemperle-Britschgi C, Hoffmann GF, Blau N, and Thöny B
- Abstract
Background: Autosomal recessive mutations in DNAJC12 , encoding a cochaperone of HSP70 with hitherto unknown function, were recently described to lead to hyperphenylalaninemia, central monoamine neurotransmitter (dopamine and serotonin) deficiency, dystonia and intellectual disability in six subjects affected by homozygous variants., Objective: Patients exhibiting hyperphenylalaninemia in whom deficiencies in hepatic phenylalanine hydroxylase and tetrahydrobiopterin cofactor metabolism had been excluded were subsequently analysed for DNAJC12 variants., Methods: To analyse DNAJC12, genomic DNA from peripheral blood (Sanger sequencing), as well as quantitative messenger RNA (Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)) and protein expression (Western blot) from primary skin fibroblasts were performed., Results: We describe five additional patients from three unrelated families with homozygosity/compound heterozygosity in DNAJC12 with three novel variants: c.85delC/p.Gln29Lysfs*38, c.596G>T/p.*199Leuext*42 and c.214C>T/p.(Arg72*). In contrast to previously reported DNAJC12-deficient patients, all five cases showed a very mild neurological phenotype. In two subjects, cerebrospinal fluid and primary skin fibroblasts were analysed showing similarly low 5-hydroxyindolacetic acid and homovanillic acid concentrations but more reduced expressions of mRNA and DNAJC12 compared with previously described patients. All patients responded to tetrahydrobiopterin challenge by lowering blood phenylalanine levels., Conclusions: DNAJC12 deficiency appears to result in a more heterogeneous neurological phenotype than originally described. While early identification and institution of treatment with tetrahydrobiopterin and neurotransmitter precursors is crucial to ensure optimal neurological outcome in DNAJC12-deficient patients with a severe phenotype, optimal treatment for patients with a milder phenotype remains to be defined., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)
- Published
- 2017
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25. Mutations in the Human Argininosuccinate Synthetase (ASS1) Gene, Impact on Patients, Common Changes, and Structural Considerations.
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Diez-Fernandez C, Rüfenacht V, and Häberle J
- Subjects
- Alleles, Amino Acid Sequence, Animals, Argininosuccinate Synthase chemistry, Argininosuccinate Synthase metabolism, Citrullinemia epidemiology, Citrullinemia therapy, Disease Models, Animal, Enzyme Activation, Genetic Association Studies, Genotype, Geography, Medical, Humans, Models, Molecular, Phenotype, Position-Specific Scoring Matrices, Prenatal Diagnosis, Protein Conformation, Severity of Illness Index, Structure-Activity Relationship, Argininosuccinate Synthase genetics, Citrullinemia diagnosis, Citrullinemia genetics, Mutation
- Abstract
Citrullinemia type 1 is an autosomal recessive urea cycle disorder caused by defects in the argininosuccinate synthetase (ASS) enzyme due to mutations in ASS1 gene. An impairment of ASS function can lead to a wide spectrum of phenotypes, from life-threatening neonatal hyperammonemia to a later onset with mild symptoms, and even some asymptomatic patients exhibiting an only biochemical phenotype. The disease is panethnic. In this update, we report 137 mutations (64 of which are novel), consisting of 89 missense mutations, 19 nonsense mutations, 17 mutations that affect splicing, and 12 deletions. The change p.Gly390Arg is by far the most common mutation and is widely spread throughout the world. Other frequent mutations (p.Arg157His, p.Trp179Arg, p.Val263Met, p.Arg304Trp, p.Gly324Ser, p.Gly362Val, and p.Arg363Trp), each found in at least 12 independent families, are mainly carried by patients from the Indian subcontinent, Turkey, Germany, and Japan. To better understand the disease, we collected clinical data of >360 patients, including all published information available. This information is related to the patients' genetic background, the conservation of the mutated residues and a structural rationalization of the effect of the most frequent mutations. In addition, we review ASS regulation, animal models, diagnostic strategies, newborn screening, and treatment options., (© 2017 WILEY PERIODICALS, INC.)
- Published
- 2017
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26. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis.
- Author
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Spodenkiewicz M, Diez-Fernandez C, Rüfenacht V, Gemperle-Britschgi C, and Häberle J
- Abstract
Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism-congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition., Competing Interests: The authors declare no conflict of interest.
- Published
- 2016
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27. Defective hepatic bicarbonate production due to carbonic anhydrase VA deficiency leads to early-onset life-threatening metabolic crisis.
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Diez-Fernandez C, Rüfenacht V, Santra S, Lund AM, Santer R, Lindner M, Tangeraas T, Unsinn C, de Lonlay P, Burlina A, van Karnebeek CD, and Häberle J
- Subjects
- Acidosis metabolism, Acidosis pathology, Adolescent, Bicarbonates metabolism, Carbonic Anhydrases deficiency, Carbonic Anhydrases metabolism, Child, Child, Preschool, Humans, Hyperammonemia metabolism, Hyperammonemia pathology, Hypoglycemia genetics, Hypoglycemia metabolism, Hypoglycemia pathology, Infant, Infant, Newborn, Lactic Acid metabolism, Liver enzymology, Liver pathology, Mitochondria genetics, Mitochondria metabolism, Mitochondria pathology, Mutation, Protein Isoforms, Acidosis genetics, Carbonic Anhydrases genetics, Hyperammonemia genetics, Liver metabolism
- Abstract
Purpose: Four mitochondrial metabolic liver enzymes require bicarbonate, which is provided by the carbonic anhydrase isoforms VA (CAVA) and VB (CAVB). Defective hepatic bicarbonate production leads to a unique combination of biochemical findings: hyperammonemia, elevated lactate and ketone bodies, metabolic acidosis, hypoglycemia, and excretion of carboxylase substrates. This study aimed to test for CAVA or CAVB deficiencies in a group of 96 patients with early-onset hyperammonemia and to prove the disease-causing role of the CAVA variants found., Methods: We performed CA5A and CA5B sequencing in the described cohort and developed an expression system using insect cells, which enabled the characterization of wild-type CAVA, clinical mutations, and three variants that affect functional residues., Results: In 10 of 96 patients, mutations in CA5A were identified on both alleles but none in CA5B. Exhibiting decreased enzyme activity or thermal stability, all CAVA mutations were proven to cause disease, whereas the three variants showed no relevant effect., Conclusion: CAVA deficiency is a differential diagnosis of early-onset and life-threatening metabolic crisis, with hyperammonemia, hyperlactatemia, and ketonuria as apparently obligate signs. It seems to be more common than other rare metabolic diseases, and early identification may allow specific treatment of hyperammonemia and ultimately prevent neurologic sequelae.Genet Med 18 10, 991-1000.
- Published
- 2016
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28. Kinetic mutations in argininosuccinate synthetase deficiency: characterisation and in vitro correction by substrate supplementation.
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Diez-Fernandez C, Wellauer O, Gemperle C, Rüfenacht V, Fingerhut R, and Häberle J
- Subjects
- Argininosuccinate Synthase metabolism, Aspartic Acid metabolism, Aspartic Acid pharmacology, Catalytic Domain genetics, Citrulline metabolism, Citrullinemia drug therapy, Citrullinemia genetics, Humans, Kinetics, Mutation, Missense, Argininosuccinate Synthase genetics, Aspartic Acid therapeutic use, Citrullinemia enzymology
- Abstract
Background: Citrullinemia type 1 is an autosomal-recessive urea cycle disorder caused by mutations in the ASS1 gene and characterised by increased plasma citrulline concentrations. Of the ∼90 argininosuccinate synthetase (ASS) missense mutations reported, 21 map near the substrate (aspartate or citrulline) binding site, and thus are potential kinetic mutations whose decreased activities could be amenable to substrate supplementation. This article aims at characterising these 21 ASS mutations to prove their disease-causing role and to test substrate supplementation as a novel therapeutic approach., Methods: We used an Escherichia coli expression system to study all potentially kinetic ASS mutations. All mutant enzymes were nickel-affinity purified, their activity and kinetic parameters were measured using tandem mass spectrometry and their thermal stability using differential scanning fluorimetry. Structural rationalisation of the effects of these mutations was performed., Results: Of the characterised mutants, 13 were totally inactive while 8 exhibited decreased affinity for aspartate and citrulline. The activity of these eight kinetic mutations could be rescued to ∼10-99% of the wild-type using high l-aspartate concentrations., Conclusions: Substrate supplementation raised in vitro the activity of eight citrullinemia type 1 mutations with reduced affinity for aspartate. As a direct translation of these results to the clinics, we propose to further evaluate the use of oxaloacetate, a nitrogen-free aspartate precursor and already available medical food (anti-ageing and brain stimulating, not considered as a drug by the US Food and Drug Administration), in patients with citrullinemia type 1 with decreased aspartate affinity. Although only patients with kinetic mutations would benefit, oxaloacetate could offer a safe novel treatment., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)
- Published
- 2016
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29. Understanding N-Acetyl-L-Glutamate Synthase Deficiency: Mutational Spectrum, Impact of Clinical Mutations on Enzyme Functionality, and Structural Considerations.
- Author
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Sancho-Vaello E, Marco-Marín C, Gougeard N, Fernández-Murga L, Rüfenacht V, Mustedanagic M, Rubio V, and Häberle J
- Subjects
- Amino-Acid N-Acetyltransferase chemistry, Amino-Acid N-Acetyltransferase metabolism, Genetic Predisposition to Disease, Humans, Models, Molecular, Protein Binding, Protein Domains, Protein Stability, Amino-Acid N-Acetyltransferase genetics, Mutation, Missense, Urea Cycle Disorders, Inborn genetics
- Abstract
N-acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification of NAGS gene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. Overall, 36 NAGS mutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele. 61% of mutations are missense changes. Lack or decrease of NAGS protein is predicted for ∼1/3 of mutations. Missense mutations frequency is inhomogeneous along NAGS: null for exon 1, but six in exon 6, which reflects the paramount substrate binding/catalytic role of the C-terminal domain (GNAT domain). Correspondingly, phenotypes associated with missense mutations mapping in the GNAT domain are more severe than phenotypes of amino acid kinase domain-mapping missense mutations. Enzyme activity and stability assays with 12 mutations introduced into pure recombinant Pseudomonas aeruginosa NAGS, together with in silico structural analysis, support the pathogenic role of most NAGSD-associated mutations found. The disease-causing mechanisms appear to be, from higher to lower frequency, decreased solubility/stability, aberrant kinetics/catalysis, and altered arginine modulation., (© 2016 WILEY PERIODICALS, INC.)
- Published
- 2016
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30. Effect of Cysteamine on Mutant ASL Proteins with Cysteine for Arginine Substitutions.
- Author
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Inauen C, Rüfenacht V, Pandey AV, Hu L, Blom H, Nuoffer JM, and Häberle J
- Subjects
- Argininosuccinate Lyase chemistry, Argininosuccinate Lyase metabolism, Cell Survival drug effects, Gene Expression Regulation drug effects, HEK293 Cells, Humans, Lysine metabolism, Models, Molecular, Molecular Docking Simulation, Amino Acid Substitution drug effects, Arginine metabolism, Argininosuccinate Lyase genetics, Cysteamine pharmacology, Cysteine metabolism
- Abstract
Introduction: Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins., Objectives: We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle., Methods: In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay., Results: Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue., Conclusion: Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.
- Published
- 2016
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31. Hepatocyte Transfection in Small Pigs After Weaning by Hydrodynamic Intraportal Injection of Naked DNA/Minicircle Vectors.
- Author
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Stoller F, Schlegel A, Viecelli HM, Rüfenacht V, Cesarovic N, Viecelli C, Deplazes S, Bettschart R, Hurter K, Schmierer P, Sidler X, Kron P, Dutkowski P, Graf R, Thöny B, and Häberle J
- Subjects
- Animals, Catheterization, Hydrodynamics, Portal Vein metabolism, Swine, Transgenes, DNA administration & dosage, Genetic Vectors administration & dosage, Liver Diseases genetics, Liver Diseases therapy, Metabolic Diseases genetics, Metabolic Diseases therapy
- Abstract
Liver is an attractive organ for gene delivery in order to correct various genetic (metabolic) diseases. Hydrodynamic vein injection of naked DNA/minicircles devoid of viral or plasmid backbones was demonstrated in, for example, murine phenylketonuria to allow sustained therapeutic transduction of hepatocytes. Here we show successful hepatocyte transfusion in domestic small pigs immediately after weaning upon portal vein catheterization and hydrodynamic injection of naked DNA/minicircle vectors expressing the luciferase gene from the CMV or a liver-specific promoter. First, we established a surgical method allowing hydrodynamic portal vein pressurization up to 120 mmHg and infusion of naked DNA in pigs (n = 5) with long-term survival. No acute adverse effects such as changes in liver transaminases or signs of liver cell damage were observed. We then showed efficiency of stable hepatocyte transfection at 10 and 28 days in single experiments (n = 7) where we found that up to 60% of samples (45/75) were polymerase chain reaction (PCR)-positive for minicircle-DNA. Of these samples, 13% of the positive specimen (6/45) showed low but stable luciferase expression when driven by a liver-specific promoter, as well as appropriate copy numbers per diploid genome. In conclusion, we accomplished a safe procedure for stable transfection of liver cells upon hydrodynamic gene delivery using minicircle vectors in small pigs as a prerequisite to potentially treat infants with genetic liver diseases.
- Published
- 2015
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32. Unstable argininosuccinate lyase in variant forms of the urea cycle disorder argininosuccinic aciduria.
- Author
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Hu L, Pandey AV, Balmer C, Eggimann S, Rüfenacht V, Nuoffer JM, and Häberle J
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Argininosuccinate Lyase chemistry, Argininosuccinic Aciduria enzymology, Enzyme Stability genetics, HEK293 Cells, Humans, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, RNA Stability genetics, RNA, Messenger chemistry, RNA, Messenger genetics, Temperature, Transfection, Urea Cycle Disorders, Inborn enzymology, Urea Cycle Disorders, Inborn genetics, Argininosuccinate Lyase genetics, Argininosuccinic Aciduria genetics, Mutation, Missense
- Abstract
Loss of function of the urea cycle enzyme argininosuccinate lyase (ASL) is caused by mutations in the ASL gene leading to ASL deficiency (ASLD). ASLD has a broad clinical spectrum ranging from life-threatening severe neonatal to asymptomatic forms. Different levels of residual ASL activity probably contribute to the phenotypic variability but reliable expression systems allowing clinically useful conclusions are not yet available. In order to define the molecular characteristics underlying the phenotypic variability, we investigated all ASL mutations that were hitherto identified in patients with late onset or mild clinical and biochemical courses by ASL expression in human embryonic kidney 293 T cells. We found residual activities >3% of ASL wild type (WT) in nine of 11 ASL mutations. Six ASL mutations (p.Arg95Cys, p.Ile100Thr, p.Val178Met, p.Glu189Gly, p.Val335Leu, and p.Arg379Cys) with residual activities ≥16% of ASL WT showed no significant or less than twofold reduced Km values, but displayed thermal instability. Computational structural analysis supported the biochemical findings by revealing multiple effects including protein instability, disruption of ionic interactions and hydrogen bonds between residues in the monomeric form of the protein, and disruption of contacts between adjacent monomeric units in the ASL tetramer. These findings suggest that the clinical and biochemical course in variant forms of ASLD is associated with relevant residual levels of ASL activity as well as instability of mutant ASL proteins. Since about 30% of known ASLD genotypes are affected by mutations studied here, ASLD should be considered as a candidate for chaperone treatment to improve mutant protein stability.
- Published
- 2015
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33. Correction: Functional Characterization of the spf/ash Splicing Variation in OTC Deficiency of Mice and Man.
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Rivera-Barahona A, Sánchez-Alcudia R, Viecelli HM, Rüfenacht V, Pérez B, Ugarte M, Häberle J, Thöny B, and Desviat LR
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- 2015
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34. Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.
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Rivera-Barahona A, Sánchez-Alcudia R, Viecelli HM, Rüfenacht V, Pérez B, Ugarte M, Häberle J, Thöny B, and Desviat LR
- Subjects
- Animals, Base Sequence, Exons, Humans, Introns, Liver enzymology, Mice, Mutation, Ornithine Carbamoyltransferase metabolism, Ornithine Carbamoyltransferase Deficiency Disease enzymology, Ornithine Carbamoyltransferase Deficiency Disease metabolism, Alternative Splicing genetics, Ornithine Carbamoyltransferase genetics, Ornithine Carbamoyltransferase Deficiency Disease genetics, RNA Splice Sites genetics
- Abstract
The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations.
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- 2015
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35. Fatal hyperammonemia and carbamoyl phosphate synthetase 1 (CPS1) deficiency following high-dose chemotherapy and autologous hematopoietic stem cell transplantation.
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Laemmle A, Hahn D, Hu L, Rüfenacht V, Gautschi M, Leibundgut K, Nuoffer JM, and Häberle J
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- Antineoplastic Combined Chemotherapy Protocols administration & dosage, Antineoplastic Combined Chemotherapy Protocols pharmacology, Brain Edema etiology, Carbamoyl-Phosphate Synthase (Ammonia) genetics, Carbamoyl-Phosphate Synthase (Ammonia) metabolism, Carbamyl Phosphate metabolism, Carboplatin adverse effects, Carboplatin pharmacology, Child, Preschool, Combined Modality Therapy, Etoposide adverse effects, Etoposide pharmacology, Fatal Outcome, Glutamine blood, Hep G2 Cells, Humans, Hyperammonemia chemically induced, Liver enzymology, Liver metabolism, Male, Middle Aged, Neuroblastoma drug therapy, Ornithine Carbamoyltransferase genetics, Oxidative Phosphorylation, Antineoplastic Combined Chemotherapy Protocols adverse effects, Carbamoyl-Phosphate Synthase (Ammonia) deficiency, Hematopoietic Stem Cell Transplantation adverse effects, Hyperammonemia etiology
- Abstract
Fatal hyperammonemia secondary to chemotherapy for hematological malignancies or following bone marrow transplantation has been described in few patients so far. In these, the pathogenesis of hyperammonemia remained unclear and was suggested to be multifactorial. We observed severe hyperammonemia (maximum 475 μmol/L) in a 2-year-old male patient, who underwent high-dose chemotherapy with carboplatin, etoposide and melphalan, and autologous hematopoietic stem cell transplantation for a neuroblastoma stage IV. Despite intensive care treatment, hyperammonemia persisted and the patient died due to cerebral edema. The biochemical profile with elevations of ammonia and glutamine (maximum 1757 μmol/L) suggested urea cycle dysfunction. In liver homogenates, enzymatic activity and protein expression of the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) were virtually absent. However, no mutation was found in CPS1 cDNA from liver and CPS1 mRNA expression was only slightly decreased. We therefore hypothesized that the acute onset of hyperammonemia was due to an acquired, chemotherapy-induced (posttranscriptional) CPS1 deficiency. This was further supported by in vitro experiments in HepG2 cells treated with carboplatin and etoposide showing a dose-dependent decrease in CPS1 protein expression. Due to severe hyperlactatemia, we analysed oxidative phosphorylation complexes in liver tissue and found reduced activities of complexes I and V, which suggested a more general mitochondrial dysfunction. This study adds to the understanding of chemotherapy-induced hyperammonemia as drug-induced CPS1 deficiency is suggested. Moreover, we highlight the need for urgent diagnostic and therapeutic strategies addressing a possible secondary urea cycle failure in future patients with hyperammonemia during chemotherapy and stem cell transplantation., (Copyright © 2015 Elsevier Inc. All rights reserved.)
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- 2015
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36. Citrin deficiency: A treatable cause of acute psychosis in adults.
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Bijarnia-Mahay S, Häberle J, Rüfenacht V, Shigematsu Y, Saxena R, and Verma IC
- Abstract
Citrin deficiency is an autosomal recessive genetic disorder caused by a defect in the mitochondrial aspartate/glutamate antiporter, citrin. The disorder manifests either as neonatal intra-hepatic cholestasis or occurs in adulthood with recurrent hyperammonemia and neuropsychiatric disturbances. It has a high prevalence in the East Asian population, but is actually pan-ethnic. We report the case of a 26-year-old male patient presenting with episodes of abnormal neuro-psychiatric behavior associated with hyperammonemia, who was diagnosed to be having citrin deficiency. Sequencing of the SLC25A13 gene revealed two novel mutations, a single base pair deletion, c. 650delT (p.Phe217SerfsFNx0133) in exon 7, and a missense mutation, c. 869T>C (p.Ile290Thr) in exon 9. Confirmation of the diagnosis allowed establishment of the appropriate management. The latter is an essential pre-requisite for obtaining a good prognosis as well as for family counseling.
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- 2015
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37. Recurrence of carbamoyl phosphate synthetase 1 (CPS1) deficiency in Turkish patients: characterization of a founder mutation by use of recombinant CPS1 from insect cells expression.
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Hu L, Diez-Fernandez C, Rüfenacht V, Hismi BÖ, Ünal Ö, Soyucen E, Çoker M, Bayraktar BT, Gunduz M, Kiykim E, Olgac A, Pérez-Tur J, Rubio V, and Häberle J
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- Animals, Carbamoyl-Phosphate Synthase (Ammonia) chemistry, Carbamoyl-Phosphate Synthase (Ammonia) metabolism, Enzyme Stability, Female, Founder Effect, Humans, Infant, Newborn, Male, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sf9 Cells, Spodoptera, Turkey, Carbamoyl-Phosphate Synthase (Ammonia) genetics, Carbamoyl-Phosphate Synthase I Deficiency Disease genetics, Sequence Deletion
- Abstract
Carbamoyl phosphate synthetase 1 (CPS1) deficiency due to CPS1 mutations is a rare autosomal-recessive urea cycle disorder causing hyperammonemia that can lead to death or severe neurological impairment. CPS1 catalyzes carbamoyl phosphate formation from ammonia, bicarbonate and two molecules of ATP, and requires the allosteric activator N-acetyl-L-glutamate. Clinical mutations occur in the entire CPS1 coding region, but mainly in single families, with little recurrence. We characterized here the only currently known recurrent CPS1 mutation, p.Val1013del, found in eleven unrelated patients of Turkish descent using recombinant His-tagged wild type or mutant CPS1 expressed in baculovirus/insect cell system. The global CPS1 reaction and the ATPase and ATP synthesis partial reactions that reflect, respectively, the bicarbonate and the carbamate phosphorylation steps, were assayed. We found that CPS1 wild type and V1013del mutant showed comparable expression levels and purity but the mutant CPS1 exhibited no significant residual activities. In the CPS1 structural model, V1013 belongs to a highly hydrophobic β-strand at the middle of the central β-sheet of the A subdomain of the carbamate phosphorylation domain and is close to the predicted carbamate tunnel that links both phosphorylation sites. Haplotype studies suggested that p.Val1013del is a founder mutation. In conclusion, the mutation p.V1013del inactivates CPS1 but does not render the enzyme grossly unstable or insoluble. Recurrence of this particular mutation in Turkish patients is likely due to a founder effect, which is consistent with the frequent consanguinity observed in the affected population., (Copyright © 2014 Elsevier Inc. All rights reserved.)
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- 2014
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38. Autism spectrum disorder associated with low serotonin in CSF and mutations in the SLC29A4 plasma membrane monoamine transporter (PMAT) gene.
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Adamsen D, Ramaekers V, Ho HT, Britschgi C, Rüfenacht V, Meili D, Bobrowski E, Philippe P, Nava C, Van Maldergem L, Bruggmann R, Walitza S, Wang J, Grünblatt E, and Thöny B
- Abstract
Background: Patients with autism spectrum disorder (ASD) may have low brain serotonin concentrations as reflected by the serotonin end-metabolite 5-hydroxyindolacetic acid (5HIAA) in cerebrospinal fluid (CSF)., Methods: We sequenced the candidate genes SLC6A4 (SERT), SLC29A4 (PMAT), and GCHFR (GFRP), followed by whole exome analysis., Results: The known heterozygous p.Gly56Ala mutation in the SLC6A4 gene was equally found in the ASD and control populations. Using a genetic candidate gene approach, we identified, in 8 patients of a cohort of 248 with ASD, a high prevalence (3.2%) of three novel heterozygous non-synonymous mutations within the SLC29A4 plasma membrane monoamine transporter (PMAT) gene, c.86A > G (p.Asp29Gly) in two patients, c.412G > A (p.Ala138Thr) in five patients, and c.978 T > G (p.Asp326Glu) in one patient. Genome analysis of unaffected parents confirmed that these PMAT mutations were not de novo but inherited mutations. Upon analyzing over 15,000 normal control chromosomes, only SLC29A4 c.86A > G was found in 23 alleles (0.14%), while neither c.412G > A (<0.007%) nor c.978 T > G (<0.007%) were observed in all chromosomes analyzed, emphasizing the rareness of the three alterations. Expression of mutations PMAT-p.Ala138Thr and p.Asp326Glu in cellulae revealed significant reduced transport uptake activity towards a variety of substrates including serotonin, dopamine, and 1-methyl-4-phenylpyridinium (MPP(+)), while mutation p.Asp29Gly had reduced transport activity only towards MPP(+). At least two ASD subjects with either the PMAT-Ala138Thr or the PMAT-Asp326Glu mutation with altered serotonin transport activity had, besides low 5HIAA in CSF, elevated serotonin levels in blood and platelets. Moreover, whole exome sequencing revealed additional alterations in these two ASD patients in mainly serotonin-homeostasis genes compared to their non-affected family members., Conclusions: Our findings link mutations in SLC29A4 to the ASD population although not invariably to low brain serotonin. PMAT dysfunction is speculated to raise serotonin prenatally, exerting a negative feedback inhibition through serotonin receptors on development of serotonin networks and local serotonin synthesis. Exome sequencing of serotonin homeostasis genes in two families illustrated more insight in aberrant serotonin signaling in ASD.
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- 2014
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39. Mutations and polymorphisms in the human argininosuccinate lyase (ASL) gene.
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Balmer C, Pandey AV, Rüfenacht V, Nuoffer JM, Fang P, Wong LJ, and Häberle J
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- Argininosuccinic Aciduria ethnology, Binding Sites, Codon, Nonsense, Genotype, Humans, Models, Molecular, Mutation, Missense, Polymorphism, Single Nucleotide, Protein Conformation, Protein Structure, Quaternary, Protein Structure, Secondary, Argininosuccinate Lyase chemistry, Argininosuccinate Lyase genetics, Argininosuccinic Aciduria genetics, Genetic Variation
- Abstract
Argininosuccinate lyase deficiency (ASLD) is caused by a defect of the urea cycle enzyme argininosuccinate lyase (ASL) encoded by the ASL gene. Patients often present early after birth with hyperammonemia but can also manifest outside the neonatal period mainly triggered by excessive protein catabolism. Clinical courses comprise asymptomatic individuals who only excrete the biochemical marker, argininosuccinic acid, in urine, and patients who succumb to their first hyperammonemic decompensation. Some patients without any hyperammonemia develop severe neurological disease. Here, we are providing an update on the molecular basis of ASLD by collecting all published (n = 67) as well as novel mutations (n = 67) of the ASL gene. We compile data on all 160 different genotypes ever identified in 223 ASLD patients, including clinical courses whenever available. Finally, we are presenting structural considerations focusing on the relevance of mutations for ASL homotetramer formation. ASLD can be considered as a panethnic disease with only single founder mutations identified in the Finnish (c.299T>C, p.Ile100Thr) and Arab (c.1060C>T, p.Gln354*) population. Most mutations are private with only few genotypes recurring in unrelated patients. The majority of mutations are missense changes including some with more frequent occurrence such as p.Arg12Gln, p.Ile100Thr, p.Val178Met, p.Arg186Trp, p.Glu189Gly, p.Gln286Arg, and p.Arg385Cys., (© 2013 WILEY PERIODICALS, INC.)
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- 2014
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40. Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria.
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Hu L, Pandey AV, Eggimann S, Rüfenacht V, Möslinger D, Nuoffer JM, and Häberle J
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- Adult, Argininosuccinate Lyase metabolism, Argininosuccinic Aciduria enzymology, Argininosuccinic Aciduria pathology, Child, Exons, Gene Expression Regulation, Genotype, HEK293 Cells, Humans, Male, Mutation, Phenotype, Protein Isoforms metabolism, Protein Structure, Quaternary, Recombinant Proteins genetics, Argininosuccinate Lyase chemistry, Argininosuccinate Lyase genetics, Argininosuccinic Aciduria genetics, Protein Isoforms chemistry, Protein Isoforms genetics
- Abstract
Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.
- Published
- 2013
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