Development of a rapid functional assay that predicts GLUT1 disease severity

摘要

Objective To examine the genotype to phenotype connection in glucose transporter type 1 (GLUT1) deficiency and whether a simple functional assay can predict disease outcome from genetic sequence alone. Methods GLUT1 deficiency, due to mutations in SLC2A1 , causes a wide range of epilepsies. One possible mechanism for this is variable impact of mutations on GLUT1 function. To test this, we measured glucose transport by GLUT1 variants identified in population controls and patients with mild to severe epilepsies. Controls were reference sequence from the NCBI and 4 population missense variants chosen from public reference control databases. Nine variants associated with epilepsies or movement disorders, with normal intellect in all individuals, formed the mild group. The severe group included 5 missense variants associated with classical GLUT1 encephalopathy. GLUT1 variants were expressed in Xenopus laevis oocytes, and glucose uptake was measured to determine kinetics (Vmax) and affinity (Km). Results Disease severity inversely correlated with rate of glucose transport between control (Vmax = 28 ± 5), mild (Vmax = 16 ± 3), and severe (Vmax = 3 ± 1) groups, respectively. Affinities of glucose binding in control (Km = 55 ± 18) and mild (Km = 43 ± 10) groups were not significantly different, whereas affinity was indeterminate in the severe group because of low transport rates. Simplified analysis of glucose transport at high concentration (100 mM) was equally effective at separating the groups. Conclusions Disease severity can be partly explained by the extent of GLUT1 dysfunction. This simple Xenopus oocyte assay complements genetic and clinical assessments. In prenatal diagnosis, this simple oocyte glucose uptake assay could be useful because standard clinical assessments are not available. Failure of the glucose transporter type 1 (GLUT1), coded by the gene Solute Carrier Family 2 member 1 ( SLC2A1) , leads to inadequate brain glucose and neurologic disorders. 1 Classical GLUT1 encephalopathy shows intractable infantile seizures, complex motor disorder, intellectual impairment, low CSF glucose (hypoglycorrhachia), and often microcephaly. 2 However, the spectrum of GLUT1 deficiency syndrome is much broader. Familial cases frequently have a combination of epilepsies with absence seizures, normal intellect, and the movement disorder of paroxysmal exertional dyskinesia. 3 GLUT1 deficiency has been described as occurring in 10% of early-onset absence epilepsy, 5% of epilepsy with myoclonic-atonic seizures, and approximately 1% of genetic generalized epilepsies. 4 , – 7 Focal epilepsies also occur. 3 , – 8 One possible mechanism for the wide range of phenotypic severity in GLUT1 deficiency syndrome is the extent to which mutations affect GLUT1 function. Deletions and null mutations of SLC2A1 are associated with severe encephalopathy, whereas missense mutations can be seen across the whole spectrum of severity. 2 , 3 , 5 , 6 , 9 , – 12 Deletions lead to complete haploinsufficiency, whereas the effects of missense changes are presumed to range from hypomorphic to complete loss of function. In this study, we examine that the range of residual function in missense variants causes GLUT1 deficiency. We compare the function of control missense SLC2A1 variants with patient variants associated with either mild disease or classical GLUT1 encephalopathy. We hypothesize that residual function of the missense alleles will be greater in those with mild disease compared with those with severe disease and that the difference will be sufficiently marked to be clinically useful in predictive testing.