Phenotype variability of GLUT1 deficiency syndrome: Description of a case series with novel SLC2A1 gene mutations

Epilepsy & Behavior 79 (2018) 169–173 Contents lists available at ScienceDirect Epilepsy & Behavior journal homepage: www.elsevier.com/locate/yebeh Brief Communication Phenotype variability of GLUT1 deficiency syndrome: Description of a case series with novel SLC2A1 gene mutations Lidia Di Vito a, Laura Licchetta a,b, Tommaso Pippucci c, Sara Baldassari a, Carlotta Stipa a,b, Barbara Mostacci a, Lara Alvisi a,b, Paolo Tinuper a,b, Francesca Bisulli a,b,⁎ a b c IRCCS Institute of Neurological Sciences, Via Altura 3, 40137 Bologna, Italy Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy U.O. Medical Genetics, Sant'Orsola-Malpighi University Hospital, Bologna, Italy a r t i c l e i n f o Article history: Received 28 September 2017 Revised 13 December 2017 Accepted 15 December 2017 Available online xxxx Keywords: Glucose transporter type I deficiency syndrome Epilepsy SLC2A1 mutation Nonepileptic paroxysmal phenomena a b s t r a c t Glucose transporter type 1 (GLUT1) deficiency due to SLC2A1 mutations causes a wide spectrum of neurologic disorders ranging from severe encephalopathy with developmental delay, epilepsy, ataxia, and acquired microcephaly to atypical less severe variants. Early diagnosis is crucial for prompt initiation of a ketogenic diet. Recognizing GLUT1 deficiency syndrome (GLUT1DS) may be challenging and results in delayed diagnosis. Here we describe the clinical and molecular findings of patients with SLC2A1 mutations referred to our adult Epilepsy Center. Patients with a clinical history suggestive of GLUT1DS were screened for SLC2A1 mutations. Blood samples were collected from probands and first-degree relatives. A lumbar puncture was performed in two patients in fasting state, and cerebrospinal fluid and blood glucose measurement were undertaken at the same time. Since 2010, 19 GLUT1DS probands have been screened for SLC2A1 mutations. We identified four different SLC2A1 mutations in three sporadic cases and one family. Three mutations (c.130_135delTACAAC, c.342_343insA, and c.845ANG) were novel, whereas one was previously reported in the literature associated with a different phenotype (c.497_499delTCG). Here we describe a small case series of patients with sporadic and familial GLUT1DS presenting with a broad phenotypic heterogeneity which is likely to be responsible for the considerable delay in diagnosis. © 2017 Elsevier Inc. All rights reserved. 1. Introduction Glucose transporter type I deficiency syndrome (GLUT1DS) is caused by a defect of glucose uptake mediated by GLUT1 at the blood–brain barrier and in the brain cells [1]. The classic phenotype is early onset encephalopathy manifesting with a range of complex movement disorders, epilepsy, intellectual disability, and acquired microcephaly [2]. Paroxysmal exercise-induced dyskinesia (PED) with or without seizures, paroxysmal ataxia, dystonia, and migraine have been recognized as atypical variants of GLUT1DS [3]. The epilepsy phenotype may vary with most patients having more than one seizure type [4]. The laboratory hallmark of GLUT1DS is a low cerebrospinal fluid (CSF) glucose concentration, i.e., b60 mg/dL or 3.3 mmol/L in all cases reported to date; b 40 mg/dL or 2.2 mmol/L in most cases [5]. ⁎ Corresponding author at: Via Altura 3, Bologna, Italy. E-mail addresses: laura.licchetta2@unibo.it (L. Licchetta), tommaso.pippucci@unibo.it (T. Pippucci), sara.baldassari@studio.unibo.it (S. Baldassari), b.mostacci@isnb.it (B. Mostacci), lara.alvisi@unibo.it (L. Alvisi), paolo.tinuper@unibo.it (P. Tinuper), francesca.bisulli@unibo.it (F. Bisulli). https://doi.org/10.1016/j.yebeh.2017.12.012 1525-5050/© 2017 Elsevier Inc. All rights reserved. SLC2A1 is the only gene associated with GLUT1DS, most SLC2A1 mutations occurring de novo. Familial cases usually show an autosomal dominant (AD) inheritance with complete penetrance, whereas autosomal recessive transmission has seldom been reported [6,7]. To date, more than 140 different mutations have been described, all leading to either complete or partial loss of function of one of the SLC2A1 alleles [2]. Missense mutations are often associated with milder symptoms, but no clear-cut phenotype–genotype correlations have been established [8]. We provide an in-depth clinical and genetic characterization of sporadic and familial cases of GLUT1DS confirmed by molecular analysis. 2. Materials and methods Since 2010, 19 patients with a clinical history suggestive of GLUT1DS have been referred to the Adult Epilepsy Center of our Institute. Clinical suspicion was based on the presence of epileptic encephalopathy or refractory epilepsy and/or movement disorders and migraine. All patients underwent a full clinical, neuroradiological (3T-brain MRI), neurophysiological examination, and neuropsychological assessment. 170 L. Di Vito et al. / Epilepsy & Behavior 79 (2018) 169–173 A lumbar puncture (LP) was performed in two patients after 6 h of fasting, and CSF and blood glucose measurements were undertaken concurrently. The blood sample was obtained before LP to avoid stress-related hyperglycemia. A CSF-to-blood glucose ratio below 0.6 was considered indicative of GLUT1DS [3]. Blood samples were collected from probands and their first-degree relatives whenever possible. SLC2A1 was analyzed partly by Sanger sequencing and multiplex ligation-dependent probe amplification and partly by a next generation sequencing (NGS) panel of genes including SCL2A1. 3. Results 3.1. Case 1 family The proband is a 20-year-old male who presented with convulsive seizures occurring in apyrexia many times per year at eight months of age. Between four and five years of age, he experienced sporadic absences followed by a seizure-free period of six years. At 11 years of age, he started having episodes of upper limb myoclonic jerks and head drop with preserved awareness. At 13 years, he had almost continuous myoclonic face and upper limb jerks associated with psychomotor slowing and frequent epileptiform abnormalities. Videoelectroencephalography (EEG) monitoring performed over the years documented a wide variability with periods of normalization followed by sudden worsening of the EEG pattern, irrespective of therapeutic changes (Fig. 1). SLC2A1 analysis (NM_006516.2) showed a heterozygous in-frame nucleotidic deletion (c.130_135del; p.Tyr44_Asn45del) inherited from the mother who has a mild intellectual disability (ID; IQ 76) and had a single febrile seizure (FS) at one year of age. Ketogenic diet (KD) started at 15 years was soon discontinued due to poor compliance. The patient still has daily seizures facilitated by fasting. 3.2. Case 2 This 21-year-old female has experienced frequent absence seizures since six months of age, facilitated by fasting and fatigue; EEG at six years of age showed diffuse, irregular spike–wave discharges at 3–4 Hz precipitated by hyperventilation. She was started on valproate (VPA) and clonazepam (CZP) with seizure control until nine years of age when seizures relapsed after antiepileptic drug (AED) withdrawal. Since 17 years of age, she has been seizure-free on low doses of lamotrigine (LTG). The patient also reported sporadic attacks of exercise-induced left leg dystonia since childhood. SLC2A1 analysis revealed a de novo heterozygous nucleotide deletion c.497_499del (p.Val166del). 3.3. Case 3 This 24-year-old male presented with a tonic seizure lasting 10–15 min at the age of six months. At the age of eight months, he started having clusters of limb spasms lasting a few seconds exacerbated by prolonged fasting. At the age of 18 months, he was started on VPA and CZP with seizure control and mild cognitive improvement. At the age of seven years, he again started having weekly spasms followed by slow falls. He has been seizure-free for the last five years on VPA and LTG. Since two years of age, he has had right leg dystonia occurring in case of fasting or prolonged exercise. SLC2A1 analysis revealed a novel nucleotide insertion (c.342_343insA) which is predicted to cause premature termination of protein synthesis (p.Leu115Thrfs*6). 3.4. Case 4 This 39-year-old female has a family history of migraine sometimes followed by transient paresis in the maternal grandfather and catamenial migraine in two maternal aunts. She presented with a prolonged Fig. 1. Case 1: Polygraphy showing spike and wave activity diffuse over both hemispheres, inconstantly associated with myoclonic jerks of the mouth and neck. FS at eight months followed by persistent left leg paralysis and psychomotor regression. Two months later, she started having monthly episodes during wakefulness characterized by head and eye rolling associated with eyelid myoclonia lasting a few minutes, and limb myoclonias. She was seizure-free for many years on sulthiame. Since the age of nine years, she has had brief episodes sometimes in clusters lasting for hours, characterized by head and eye rolling, abrupt shoulders elevation, and eyelid myoclonia. These events typically occurred on morning awakening. Ictal EEG showed diffuse polyspike–wave discharges. Since six years of age, she has also had migraine attacks associated with nausea and vomiting lasting 1 h, sometimes preceded by visual aura, and followed by worsening of left hemiparesis or right hemiparesis at least on one occasion lasting a couple of hours. The EEG recorded during the episodes showed slow background activity on the hemisphere contralateral to the hemiparesis (Fig. 2). Despite a combination of three AEDs therapy and vagus nerve stimulation, the patient has continued having multiple seizures per week with possible falls. At 39 years of age, she was started on KD, with seizure control after a six-month follow-up period. Genetic analysis, performed by NGS panel including genes associated with hemiplegic migraine (ATP1A3, ATP1 A2, CACNA1A, PRRT2 and SCN1A), identified a heterozygous missense mutation (c.845A N G; p.Gln282Arg) of SLC2A1. Additional clinical and instrumental data are listed in Table 1. L. Di Vito et al. / Epilepsy & Behavior 79 (2018) 169–173 171 Fig. 2. Case 4: EEG findings during a paroxysmal event and after resolution. (a) EEG recorded during headache associated with right hemiparesis, a paroxysmal episode and transitory aphasia. Note the marked inter-hemispheric asymmetry, with slow polymorphic theta-delta activity over the left hemisphere. (b) EEG recorded the day after the paroxysmal episode during a symptom-free phase. The inter-hemispheric asymmetry is much less evident. Note the interictal discharges of spikes and waves diffused over both hemispheres. 4. Discussion This small series of sporadic and familial cases is fully representative of the clinical spectrum of GLUT1DS due to SLC2A1 mutations. Three out the four identified pathogenic variants have never been reported. The novel p.Tyr44_Asn45del segregated with an AD pattern in two members of a small pedigree (Case 1) and is associated with low levels of glycorrhachia on lumbar puncture. Our family shows the well-known phenotypic variability among affected members ranging from mild ID and a single FS in the mother to a severe epileptic encephalopathy associated with absences seizures, myoclonic and tonic–clonic seizures, and high variability in EEG over time in the index case. Patients with variable seizure types and EEG findings, including focal or multifocal findings, may be strong candidates for GLUT1DS testing [4]. Although inherited SLC2A1 mutations are usually associated with milder manifestations than de novo mutations [9], the index case in our small cohort showed a more severe epilepsy phenotype than the other sporadic cases. The phenotype variability among affected family members sharing the same mutation suggests that other factors such as modifying genes and proteins may alter the phenotypic expression and potentially contribute to the pathophysiology of this complex disorder [10]. None of the affected members in our family had PED, which is considered one of the red flags of GLUT1DS, frequently described in familial cases [11], unlike two out of three sporadic patients who presented with movement disorders associated with fasting or exercise. In particular, Case 2 showed a typical GLUT1DS with PED and early onset absence epilepsy controlled by AED. Molecular analysis disclosed the p.Val166del mutation, already reported in a single patient whose clinical phenotype was unremarkable for epilepsy. The aminoacid Val166 is located in the transmembrane segment 5 of the GLUT1 glucose transporter; its deletion might influence the hydrophilic interaction of the water-accessible face, hindering the transport of glucose through the membrane [12]. In Case 3, PED was associated with epilepsy and ID. In this case we identified a novel mutation in the GLUT1 gene (c.342_343insA) whose pathogenic effect was predicted by different tools. Even though paroxysmal nonepileptic phenomena associated with GLUT1DS have been extensively described in the literature [13,14], there are only anecdotal reports of GLUT1D patients with clinical features of alternating hemiplegia of childhood (AHC), hemiplegic migraine (HM) [15,16], or overlapping symptoms of both AHC and HM [17]. One of our patients (Case 4) presented with nonepileptic paroxysmal events characterized by a variable combination of migraine, visual aura, autonomic symptoms, aphasia, and alternating hemiparesis, compatible with HM. As already reported, the paroxysmal episodes in our patient are associated with contralateral slowing of background activity on EEG recording [18]. These nonepileptic phenomena whose physiopathogenic mechanism is not fully understood expand the clinical spectrum of GLUT1DS. In case 3, SCL2A1 analysis disclosed the heterozygous missense variant c.845A N G (p.Gln282Arg), predicted to be deleterious for protein function (according to Polyphen2, Sift and CAAD scores), affecting a conserved amino acid residue. This mutation has never been reported in the literature, but the p.Q282_S285del deletion has been described [19,20] suggesting that the affected and surrounding amino acid residue may be important for SLC2A1 function in relation to GLUT1DS. Segregation analysis revealed that the variant is absent in the patient's healthy mother, reinforcing its pathogenic role, although the segregation in the healthy father could not be assessed. All of our patients underwent a long diagnostic workup, and consequently a long delay (mean 22.7 years) in diagnosing GLUT1DS. They were all diagnosed as adults and therefore could not benefit from KD in the early stages of the disease which may have contributed to the worsening of the clinical manifestation. Early treatment of GLUT1DS with KD is important since it provides an alternative source of energy for the brain possibly improving the long-term neurologic outcome; KD seems to be more effective in the control of seizures [2], while its effect on motor and cognitive symptoms is more controversial. On the basis of this evidence, we proposed a KD in particular to those patients with refractory epilepsy (cases 1 and 4). Only case 4 followed a KD properly, reporting an immediate good response in terms of seizure control. Due to poor compliance, KD treatment was soon discontinued on case 1 for whom modified forms of the KD or triheptanoin supplementation could be considered [21,22]. Application of a diagnostic epilepsy NGS gene panel as a first-tier screening analysis in newborns is a promising tool to shorten the time to diagnosis in these clinically heterogeneous epilepsies. In conclusion, GLUT1DS should be considered in patients with unknown drug-resistant epilepsy and cognitive dysfunction especially when they have a family history of epilepsy or mental delay. Nonepileptic paroxysmal events in GLUT1D are clearly classifiable No (HC N10%ile at age 9 y) Case 4 F, 38 y Tongue apraxia, right limb dystonia with hands stereotypies, scoliosis. Hypoplasia and hyposthenia of the left leg Case 2 F, 21 y Case 3 M, 24 y Abbreviations: Pt: patient; HC: head circumference; CSF: cerebrospinal fluid; M: male, F: female; T–C: tonic–clonic; mth: months, y: years; MMSEc: Mini Mental State Evaluation (correct score); PED: paroxysmal exercise-induced dystonia; np: not performed; ID intellectual disability; FS: febrile seizure; IQ: intelligence quotient; VPA: valproic acid; CBZ: carbamazepine; PB: phenobarbital; LEV: levetiracetam; LTG: lamotrigine; CZP: clonazepam; RFN: rufinamide; AZM: acetazolamide; VNS: vagus nerve stimulation. Normal Hemiplegic migraine 8 mth FS Absence Myoclonic Sulthiame VPA, LEV RFN, AZM VNS 39 yrs 21 yrs glucose 36 mg/dL (50–80) CSF/plasma glucose ratio 45%. lactate: normal NP Normal PED 6 mth T–C Spasms VPA, CZP, LTG 17 yrs NP Normal No (HC not available) No (HC 50%ile at birth) Normal intelligence, Dyslexia (WISC-R at 13y) Severe ID (Stand for Binet: IQ 60 at 5 y IQ 47 at 8 y IQ 35 at 12 y) Severe/moderate ID – WISC at 9 y: unscorable; − WAIS-R, MMSE at 32 y: IQ 45, MMSEc: 23.42; − MMSEc at 39 y: 22.42 6 mth Absence VPA, CZP PED 14 yrs glucose 40 mg/dL (50–80) CSF/plasma glucose ratio b 45%. lactate: normal Normal Facial dysmorphisms, dysarthria, hypotonia, hyporeflexia, adiadochokinesia, upper limbs myoclonus, mild ataxia Normal Case 1 M, 20 y No (HC N98%ile at birth) Moderate ID stable over the time (Leiter R at 16 y: IQ 36) 8 mth T–C Absence Myoclonic VPA, CBZ, PB, LEV, LTG No Diagnosis delay Neurological examination Pt Table 1 Clinical and instrumental findings. Microcephaly (HC, %ile) Neuropsychological features Age at epilepsy onset Type of epileptic seizures Therapy Nonepileptic paroxysmal events CSF analysis L. Di Vito et al. / Epilepsy & Behavior 79 (2018) 169–173 Brain MRI 172 as movement disorders, but also others including prolonged episodes of migraine, autonomic symptoms, and motor deficits should be carefully investigated as part of the clinical variability of the syndrome. Early diagnosis and treatment are important for seizure control and to prevent cognitive dysfunction. Acknowledgments Lidia Di Vito and Laura Licchetta were involved in the conception and writing of the manuscript; Carlotta Stipa, Barbara Mostacci, Sara Baldassarri and Lara Alvisi provided critical questions and suggestions to the manuscript. Sara Baldassarri performed genetic analysis, Lara Alvisi performed neurophysiological studies. Paolo Tinuper and Tommaso Pippucci revised the manuscript critically for important intellectual content. 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