Monitoring metabolites at the point of care could improve the diagnosis and management of numerous diseases. Yet for most metabolites, such assays are not available. We introduce semisynthetic, ...light-emitting sensor proteins for use in paper-based metabolic assays. The metabolite is oxidized by nicotinamide adenine dinucleotide phosphate, and the sensor changes color in the presence of the reduced cofactor, enabling metabolite quantification with the use of a digital camera. The approach makes any metabolite that can be oxidized by the cofactor a candidate for quantitative point-of-care assays, as shown for phenylalanine, glucose, and glutamate. Phenylalanine blood levels of phenylketonuria patients were analyzed at the point of care within minutes with only 0.5 microliters of blood. Results were within 15% of those obtained with standard testing methods.
Glycogen storage disease type I (GSD I) is a rare autosomal recessive disorder of carbohydate metabolism characterized by recurrent hypoglycaemia and hepatomegaly. Management of GSD I is demanding ...and comprises a diet with defined carbohydrate intake and the use of complex carbohydrates, nocturnal tube feeding or night-time uncooked cornstarch intake, regular blood glucose monitoring and the handling of emergency situations. With improved treatment, most patients nowadays survive into adulthood. Little research has been performed on the impact of GSD I on daily life, especially in adult patients. In this multi-centre study we assessed the impact of GSD I on adult daily life in 34 GSD I patients (27 GSD Ia, 7 GSD Ib) between 17 and 54 years (median 26 years) using a self-designed questionnaire that specifically focused on different aspects of daily life, such as job situation, social life, sports, travelling, composition of the household, night-time and day-time dietary management and disease monitoring as well as the patient's attitude towards the disease. At the time of investigation, the majority of patients either attended school or university or were employed, while 3 patients (9%) were out of work. Most patients ranked GSD I as a disease with moderate severity and disease burden. Dietary treatment was considered challenging by many, but the vast majority of patients considered life with GSD I as well-manageable. Although the management of GSD I poses a significant burden on daily life, most patients live an independent adult life, have a positive attitude towards their disease and seem to cope well with their situation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Inborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone ...disease or immunodeficiency. However, the clinical presentation can be nonspecific and mild so that a number of cases may be missed. Previously published assays lacked detection of certain diagnostically important biomarkers, including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine, necessitating the use of separate assays for their detection. Moreover, the limited sensitivity for some analytes in earlier assays may have hampered the reliable detection of mild cases. Therefore, we aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine.
The assay was developed and validated using LC-MS/MS and clinically tested by analyzing ERNDIM Diagnostic Proficiency Testing (DPT) samples and further specimens from patients with various purine and pyrimidine disorders.
Reliable determination of 27 analytes including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation. The method clearly distinguished pathological and normal samples and differentiated between purine and pyrimidine defects in all clinical specimens.
A LC-MS/MS assay allowing the simultaneous, sensitive and reliable diagnosis of an extended range of purine and pyrimidine disorders has been developed. The validated method has successfully been tested using ERNDIM Diagnostic Proficiency Testing (DPT) samples and further clinical specimens from patients with various purine and pyrimidine disorders. Sample preparation is simple and assay duration is short, facilitating an easier inclusion of the assay into the diagnostic procedures.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de ...novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione ...(PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD “ene” reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Newborn screening (NBS) programs are effective measures of secondary prevention and have been successively extended. We aimed to evaluate NBS for methylmalonic acidurias, propionic acidemia, ...homocystinuria, remethylation disorders and neonatal vitamin B
deficiency, and report on the identification of cofactor-responsive disease variants. This evaluation of the previously established combined multiple-tier NBS algorithm is part of the prospective pilot study "NGS2025" from August 2016 to September 2022. In 548,707 newborns, the combined algorithm was applied and led to positive NBS results in 458 of them. Overall, 166 newborns (prevalence 1: 3305) were confirmed (positive predictive value: 0.36); specifically, methylmalonic acidurias (N = 5), propionic acidemia (N = 4), remethylation disorders (N = 4), cystathionine beta-synthase (CBS) deficiency (N = 1) and neonatal vitamin B
deficiency (N = 153). The majority of the identified newborns were asymptomatic at the time of the first NBS report (total: 161/166, inherited metabolic diseases: 9/14, vitamin B
deficiency: 153/153). Three individuals were cofactor-responsive (methylmalonic acidurias: 2, CBS deficiency: 1), and could be treated by vitamin B
, vitamin B
respectively, only. In conclusion, the combined NBS algorithm is technically feasible, allows the identification of attenuated and severe disease courses and can be considered to be evaluated for inclusion in national NBS panels.
Abnormal sterol metabolism in holoprosencephaly Haas, Dorothea; Muenke, Maximilian
American journal of medical genetics. Part C, Seminars in medical genetics,
15 February 2010, Volume:
154C, Issue:
1
Journal Article
Phenylketonuria (PKU; OMIM#261600) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene resulting in high phenylalanine (Phe) in blood and brain. If not ...treated early this results in intellectual disability, behavioral and psychiatric problems, microcephaly, motor deficits, eczematous rash, autism, seizures, and developmental problems. There is a controversial discussion of whether patients with PKU have an additional risk for atherosclerosis due to interference of Phe with cholesterol synthesis and LDL-cholesterol regulation. Since cholesterol also plays a role in membrane structure and myelination, better insight into the clinical significance of the impact of Phe on lipoprotein metabolism is desirable. In 22 treated PKU patients (mean age 38.7 years) and 14 healthy controls (mean age 35.2 years), we investigated plasma with NMR spectroscopy and quantified 105 lipoprotein parameters (including lipoprotein subclasses) and 24 low molecular weight parameters. Analysis was performed on a 600 MHz Bruker AVANCE IVDr spectrometer as previously described.
Concurrent plasma Phe in PKU patients showed a wide range with a mean of 899 μmol/L (50-1318 μmol/L). Total cholesterol and LDL-cholesterol were significantly lower in PKU patients versus controls: 179.4 versus 200.9 mg/dL (p < 0.02) and 79.5 versus 104.1 mg/dL (p < 0.0038), respectively. PKU patients also had lower levels of 22 LDL subclasses with the greatest differences in LDL2 Apo-B, LDL2 Particle Number, LDL2-phospholipids, and LDL2-cholesterol (p < 0.0001). There was a slight negative correlation of total cholesterol and LDL-cholesterol with concurrent Phe level. VLDL5-free cholesterol, VLDL5-cholesterol, VLDL5-phospholipids, and VLDL4-free cholesterol showed a significant (p < 0.05) negative correlation with concurrent Phe level. There was no difference in HDL and their subclasses between PKU patients and controls. Tyrosine, glutamine, and creatinine were significantly lower in PKU patients compared to controls, while citric and glutamic acids were significantly higher.
Using NMR spectroscopy, a unique lipoprotein profile in PKU patients can be demonstrated which mimics a non-atherogenic profile as seen in patients treated by statins.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the de novo coenzyme A (CoA) synthesis starting from pantothenate. Mutations in PPCS cause autosomal-recessive dilated ...cardiomyopathy, often fatal, without apparent neurodegeneration, whereas pathogenic variants in PANK2 and COASY, two other genes involved in the CoA synthesis, cause Neurodegeneration with Brain Iron Accumulation (NBIA). PPCS-deficiency is a relatively new disease with unclear pathogenesis and no targeted therapy. Here, we report the generation of induced pluripotent stem cells from fibroblasts of two PPCS-deficient patients. These cellular models could represent a platform for pathophysiological studies and testing of therapeutic compounds for PPCS-deficiency.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
Adenosine kinase deficiency is a recently described defect affecting methionine metabolism with a severe clinical phenotype comprising mainly neurological and hepatic impairment and ...dysmorphism.
Methods
Clinical data of 11 additional patients from eight families with adenosine kinase deficiency were gathered through a retrospective questionnaire. Two liver biopsies of one patient were systematically evaluated.
Results
The main clinical symptoms are mild to severe liver dysfunction with neonatal onset, muscular hypotonia, global developmental retardation and dysmorphism (especially frontal bossing). Hepatic involvement is not a constant finding. Most patients have epilepsy and recurrent hypoglycemia due to hyperinsulinism. Major biochemical findings are intermittent hypermethioninemia, increased S-adenosylmethionine and S-adenosylhomocysteine in plasma and increased adenosine in urine. S-adenosylmethionine and S-adenosylhomocysteine are the most reliable biochemical markers. The major histological finding was pronounced microvesicular hepatic steatosis. Therapeutic trials with a methionine restricted diet indicate a potential beneficial effect on biochemical and clinical parameters in four patients and hyperinsulinism was responsive to diazoxide in two patients.
Conclusion
Adenosine kinase deficiency is a severe inborn error at the cross-road of methionine and adenosine metabolism that mainly causes dysmorphism, brain and liver symptoms, but also recurrent hypoglycemia. The clinical phenotype varies from an exclusively neurological to a multi-organ manifestation. Methionine-restricted diet should be considered as a therapeutic option.
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FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
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