Abstract
Four unrelated patients with glyceroluria ranging from 7 to 170 mmol/l were studied. The activity of glycerol kinase (GK) in cultured fibroblasts was determined with a specific enzyme assay and with two indirect methods, that is, incorporation into macromolecules of [14C] from [14C]glycerol and its oxidation to [14C]CO2. Exon amplification and RT-PCR were used to identify mutations. In patient 1, with low activity in all three assays, we identified a c.1194A>C (E398D) missense mutation. In patient 2 with a considerable activity of the GK enzyme (22% of reference), oxidation to [14C]CO2 (37%) and a high incorporation of [14C] into macromolecules (92%), we identified a c.182T>C (L61P) mutation that causes the enzyme to have a higher Km for glycerol (∼300 μ M) than normals (2–8 μ M). In patient 3, the GK activity estimated by the three different methods ranged from 16 to 22% of reference. Analysis of mRNA from the GK gene revealed three alternatively spliced transcripts. A mutation in intron 3 (g.16835G>A) resulted in an insertion of a cryptic exon between exon 2 or 3 and exon 4. Patient 4 with minor glyceroluria (7 mmol/l) and normal plasma glycerol concentration had normal activity with all three assay methods, thus excluding GK deficiency (GKD) as a cause of slight glyceroluria. To evaluate fully patients with glyceroluria, one needs to measure the GK activity and relate this and the clinical data to genetic findings. Residual enzyme activities in cultured fibroblasts can be found in GKD patients with severe clinical symptoms.
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Acknowledgements
DRS, pediatrician, is a staff member of the Department of Child Health, Dr Cipto Mangunkusumo National General Hospital, University of Indonesia – School of Medicine, Jakarta, Indonesia. She is recipient of a grant from the Van Deventer-Maas Foundation, The Netherlands. We acknowledge the technical assistance of Mr PHA van Zon in DNA mutation analysis, Mr VH Garritsen for [14C]glycerol incorporation studies, Dr Isabelle Vernhet (Department of Medical Genetics and Metabolic Disease, CHU Pellegrin, Bordeaux, France) and Dr Wolfgang Getzner (Zentrallabor, Universitätskliniken Innsbruck, Austria) for glycerol analysis in the urine of patients 1 and 2, respectively, and Dr James R Ellis (Division of Bioengineering and Physical Sciences, Office of Research Services, National Institute of Health, Bethesda, USA) for performing the splice site analysis.
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Sjarif, D., Hellerud, C., Amstel, J. et al. Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation. Eur J Hum Genet 12, 424–432 (2004). https://doi.org/10.1038/sj.ejhg.5201172
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DOI: https://doi.org/10.1038/sj.ejhg.5201172
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