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.1194A4C (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.182T4C (L61P) mutation that causes the enzyme to have a higher K m for glycerol (B300 lM) than normals (2 -8 lM). 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.16835G4A) 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.

A Novel Splice-Site Mutation of the Gk Gene in a Chinese Patient with Isolate Glycerol Kinase Deficiency

“…The mutation created a cryptic splice acceptor site, resulting in alternative splicing of the transcript with a small amount of correctly spliced GK mRNA. Likewise, we found that there were normal splicing in the splice-site mutations reported previous [4,5]. Sequencing the fragments, we found that the mutation caused a skipping of the whole exon17, leading a frame shift mutant of p. Val446Glufs*48.…”

“…Zhang et al, found that aberrant mRNA generated by splice site mutations of the GK gene can be eliminated by NMD in the asymptomatic form of GKD [9]. In contrary, there was no NMD in our study, and reviewed the other split-site mutations previously reported, no NMD was found too [4,5]. Fourth, some studies demonstrated that glycerolutilization by the cells (especially, hepatocytes) is rate-limited by the membrane permeation step, rather than the GK step [13][14][15].…”

“…Interestingly, most of the patients with split-site mutation reported previously presented with asymptomatic form of GKD [4,5,[7][8][9]. Only one patient from the literature (IVS9-1G>T) who had a history of metabolic and CNS crises at the age 6 years and 3 months, indicating the symptomatic form of GKD, but he was uneventful in the neonatal period and early childhood [10].…”

Glycerol loss to water exceeds glycerol catabolism via glycerol kinase in freeze-resistant rainbow smelt (Osmerus mordax)

“…1). In mammals, normal plasma glycerol levels range between 0.04 and 0.4 mM (18) with higher levels reported in cases subject to GK deficiency (32). Glycerol levels generally reflect the state of fat mobilization.…”

Hypoglycemia as a Pathological Result in Medical Praxis

“…GK deficiency (GKD) is an X-linked inborn error of metabolism that is characterized biochemically by hyperglycerolemia and glyceroluria and is due to mutations within or deletions of the GK gene on Xp21 (Rahib et al, 2007). Isolated GKD can occur in patients with or without symptoms, mainly due to disturbed energy homeostasis associated with hyperketotic hypoglycemia (Sjarif et al, 2004). The greater importance of glycerol as a gluconeogenetic substrate in children than in adults may explain the episodes in young patients with GKD, often elicited by catabolic stress .…”