title: Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation.
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 [(14)C] from [(14)C]glycerol and its oxidation to [(14)C]CO(2). 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 [(14)C]CO(2) (37%) and a high incorporation of [(14)C] into macromolecules (92%), we identified a c.182T>C (L61P) mutation that causes the enzyme to have a higher K(m) for glycerol ( approximately 300 microM) than normals (2-8 microM). 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.
authors.affiliation: Department of Medical Genetics, University Medical Center, Utrecht, The Netherlands.
authors.forename: Damayanti R
authors.initials: DR
authors.lastname: Sjarif
authors.forename: Christina
authors.initials: C
authors.lastname: Hellerud
authors.forename: Johannes K Ploos
authors.initials: JK
authors.lastname: van Amstel
authors.forename: Willem J
authors.initials: WJ
authors.lastname: Kleijer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sperl
authors.forename: Didier
authors.initials: D
authors.lastname: Lacombe
authors.forename: Jõrn Oliver
authors.initials: JO
authors.lastname: Sass
authors.forename: Frits A
authors.initials: FA
authors.lastname: Beemer
authors.forename: Marinus
authors.initials: M
authors.lastname: Duran
authors.forename: Bwee Tien
authors.initials: BT
authors.lastname: Poll-The
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D002648:Child; D002675:Child, Preschool; D005091:Exons / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D005990:Glycerol / Q000378:metabolism; D005991:Glycerol Kinase / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D007438:Introns / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D005991:Glycerol Kinase; D005990:Glycerol