Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation.
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.
Sjarif
Damayanti R
DR
Department of Medical Genetics, University Medical Center, Utrecht, The Netherlands.
Hellerud
Christina
C
van Amstel
Johannes K Ploos
JK
Kleijer
Willem J
WJ
Sperl
Wolfgang
W
Lacombe
Didier
D
Sass
Jõrn Oliver
JO
Beemer
Frits A
FA
Duran
Marinus
M
Poll-The
Bwee Tien
BT
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
D012333:RNA, Messenger; D005991:Glycerol Kinase; D005990:Glycerol