Prospective Multicenter Validation of a Simple Blood Test for the Diagnosis of Glut1 Deficiency Syndrome

Abstract

Background and objective: GLUT1 deficiency syndrome (Glut1DS) is a treatable neurometabolic disease that causes a wide range of neurologic symptoms in children and adults. However, its diagnosis relies on an invasive test, that is, a lumbar puncture (LP) to measure glycorrhachia, and sometimes complex molecular analyses of the SLC2A1 gene. This procedure limits the number of patients able to receive the standard of care. We wished to validate the diagnostic performance of METAglut1, a simple blood test that quantifies GLUT1 on the erythrocyte surface.

Methods: We performed a multicenter validation study in France, involving 33 centers. We studied 2 patient cohorts: a prospective cohort consisting of patients with a clinical suspicion of Glut1DS explored through the reference strategy, that is, LP and analyses of the SLC2A1 gene, and a retrospective cohort that included patients previously diagnosed with Glut1DS. All patients were blind-tested with METAglut1.

Results: We analyzed 428 patients in the prospective cohort, including 15 patients newly diagnosed with Glut1DS, and 67 patients in the retrospective cohort. METAglut1 was 80% sensitive and >99% specific for the diagnosis of Glut1DS. Concordance analyses showed a substantial agreement between METAglut1 and glycorrhachia. In the prospective cohort, the positive predictive value of METAglut1 was slightly higher than that of glycorrhachia. METAglut1 succeeded to identify patients with Glut1DS with SCL2A1 mosaicism and variants of unknown significance.

Discussion: METAglut1 is an easily performed, robust, and noninvasive diagnostic test for the diagnosis of Glut1DS, which allows wide screening of children and adults, including those with atypical forms of this treatable condition.

Classification of evidence: This study provides Class I evidence that a positive METAglut1 test accurately distinguishes patients with suspected GLUT1 deficiency syndrome from other neurologic syndromes as compared with invasive and genetic testing.

Figure 1. Patient Groups Used for the Calculation of Diagnostic Performance

We enrolled 549 patients in the prospective cohort and 87 patients in the retrospective cohort. Within the prospective cohort, 121 participants had to be excluded from the analyses, mainly because METAglut1 was the only available test for these patients (n = 116), with neither available glycorrhachia (lumbar puncture refused by the patients or their caregivers) nor molecular analyses, or because the final diagnosis remained uncertain (n = 5). After excluding mainly patients for whom molecular analyses were not available (n = 218) and a few familial cases (n = 5), 205 index patients were used to compute the sensitivity, specificity, and positive and negative predictive values of METAglut1. These performance criteria were also computed on 350 prospective patients with available glycorrhachia, after excluding 5 familial cases and 10 patients for whom blinding was not maintained—for example, patients for whom glycorrhachia was performed as a confirmatory test after METAglut1 or molecular analyses. Concordance analyses between METAglut1 and glycorrhachia were performed on this same subgroup of patients. Within the retrospective cohort, 10 patients were excluded because of previous enrollment in the initial cohort (n = 5), inconclusive or missing data (n = 1), or uncertain Glut1DS diagnosis (n = 4). From the 77 remaining patients, 60 index patients with Glut1DS were used for sensitivity analysis, after excluding 7 familial cases and revised Glut1DS diagnosis (n = 10). The 54 index patients for whom glycorrhachia was available from the retrospective cohort were used for concordance analyses between METAglut1 and glycorrhachia, after merging them with the prospective cohort of 350 index patients. PPV = positive predictive value; NPV = negative predictive value.

Figure 2. Identification of a Case of Genetic Mosaicism With METAglut1

A 12-year-old adolescent patient presented with a moderate phenotype (myoclonic epilepsy, attention, and executive function deficit) and a glycorrhachia of 2.6 mM (47 mg/dL). His red blood cells analyzed with METAglut1 showed an unusual feature with 2 distinct red blood cell populations. One population representing 36% of the patient red blood cells had a normal expression of GLUT1 while the other representing 64% of red blood cells had a distinct lower level of GLUT1 (53% of the controls). Parents of the child were healthy, with normal GLUT1 levels on red blood cells. On deeper sequencing analysis, it was confirmed that 36% of SLC2A1 copies in the patient's blood DNA and 22% in his oral swab DNA harbored a de novo premature stop codon in the SCL2A1 gene (pArg330*).

Figure 3. Glucose Uptake Assay

Glucose uptake assay by red blood cells was performed on METAglut1 false-negative patients. The glucose uptake assay interpretation threshold was set at 74% of controls. In 12 index patients with Glut1DS, 4 turned out to have an abnormally low glucose uptake, whereas 5 displayed a normal glucose uptake and 3 an abnormally high glucose uptake.

Figure 4. Concordance Analysis Between Glycorrhachia and METAglut1

All patients with both glycorrhachia and METAglut1 available at the time of completion of the study were used to draw the comparative distribution of the 2 biomarkers. The recommended interpretation thresholds are represented with dashed lines at 2.2 mM (40 mg/dL) for glycorrhachia and 76% of normal expression for METAglut1. The Cohen kappa coefficient was computed on the prospective cohort (n = 350 patients)and the whole cohort—350 prospective patients and 48 retrospective patients—for whom both glycorrhachia and METAglut1 were available.

Figure 5. Proposed New Diagnostic Algorithm for Glut1DS Diagnosis in the Standard of Care

Positive and negative predictive values of METAglut1 are those obtained from the prospective cohort, with all patients with confirmed Glut1DS, whether they had glycorrhachia available. Glut1DS = Glut1 deficiency syndrome; DEE = developmental and epileptic encephalopathy; Se = sensitivity; Spe = Specificity; PPV = positive predictive value; NPV = negative predictive value; CSF = cerebrospinal fluid.