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Pediatr Diabetes. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Pediatr Diabetes. 2016 December ; 17(8): 617–622. doi:10.1111/pedi.12346.
Epilepsy and behavioral changes, type 1 diabetes mellitus and a high titer of glutamic acid decarboxylase antibodies Esther Ganelin-Cohena,b, Dalit Modan-Mosesc,b, Rina Hemid, Hannah Kanetyd, Bruria Benzeeva,b,†, and Christiane S. Hampee,† aPediatric
Neurology Unit, The Edmond and Lily Safra Children’s Hospital, Chaim Sheba Medical Center, Ramat-Gan, Israel
Author Manuscript
bThe
Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel
cPediatric
Endocrinology and Diabetes Unit, The Edmond and Lily Safra Children’s Hospital, Chaim Sheba Medical Center, Ramat-Gan, Israel
dInstitute
of Endocrinology, Chaim Sheba Medical Center, Ramat-Gan, Israel
eDepartment
of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA, USA
Abstract
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Autoantibodies to the 65 kDa isoform of glutamate acid decarboxylase (GAD65Ab) are associated with a range of clinical disorders, including type 1 diabetes (T1D) and stiff-person syndrome (SPS). In this article we describe a young girl who was diagnosed with T1D at the end of her first year of life and developed drug-resistant epilepsy 18 months later, followed by behavioral disturbances. She was admitted to our center at the age of 5 yr, at which time high GAD65Ab titers were detected in the patient’s serum and cerebrospinal fluid (CSF). The titer remained elevated during 19 months of follow-up. Furthermore, GAD65Ab in both serum and CSF showed epitope binding characteristics similar to those observed for GAD65Ab in SPS patients, and GAD65Ab in the serum reduced GAD65 enzyme activity. Our results suggest an association between high GAD65Ab titers and epilepsy in children with T1D. Careful titration and characterization of GAD65Ab regarding inhibition of enzyme activity and epitope specificity may be helpful in identifying T1D patients at risk for neurological complications.
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Keywords epilepsy; glutamate decarboxylase antibodies; immune-mediated; pediatric; type 1 diabetes Antibodies to the 65 kDa isoform of glutamate acid decarboxylase (GAD65Ab) are associated with several diseases, including type 1 diabetes (T1D) (80% of new onset patients), stiff-person syndrome (SPS) (60–80% of patients) (1), cerebellar ataxia (30–60%
Corresponding author: Esther Ganelin-Cohen, MD, PhD, Pediatric Neurology Unit, The Edmond and Lily Safra Children’s Hospital, Chaim Sheba Medical Center, Derech Sheba 2, Ramat-Gan, 52621, Israel. Tel: 972-35302687; 972-35302685; fax: 972-35305031; [email protected]. †Both authors contributed equally to this study.
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of patients) (2), intractable epilepsy (10–30% of patients) (for review see reference 3), and Batten disease (100% of patients) (4). Presence of GAD65Ab may predict development of neurological disorders as the antibodies may already be found in preclinical stages (5, 6).
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GAD65Ab in patients with neurological manifestation differ from those in T1D patients as manifested in titers exceeding those in T1D by at least 100-fold (7, 8). In addition, GAD65Ab from patients with neurological symptoms recognize both linear and conformational epitopes, while GAD65Ab from T1D patients without neurological complications only recognize conformational epitopes. (5), suggesting that the diseasespecific immune responses are reflected in GAD65Ab characteristics (9, 10). GAD65Ab in neurological diseases may have a pathological role mediated by antibody-induced inhibition of GAD65 enzyme activity (11). It is noteworthy that Gamma-Aminobutyric Acid (GABA) levels in the patients are often reduced (12). Furthermore, monoclonal GAD65Ab representing GAD65Ab specificities in neurological conditions lead to functional impairment of GABAergic synaptic transmission both in vitro and in vivo, while monoclonal GAD65Ab representing GAD65Ab specificities in T1D do not have these effects (13, 14). In this article we describe a case of a girl diagnosed with T1D, who presented with neurological manifestations and very highGAD65Ab titers. We characterized her GAD65Ab in serum and cerebrospinal fluid (CSF) regarding titer, epitope specificity, and inhibition of GAD65 enzyme activity.
Patient presentation Author Manuscript
A 5-yr-old girl with T1D was referred to our clinic for re-evaluation of intractable epilepsy. She was the youngest of five healthy siblings, born to healthy non-consanguineous Arab parents. The pregnancy was uneventful. During the first year of life she achieved normal developmental milestones. She was diagnosed with T1D at the age of 1 yr and was treated with insulin. Epileptic seizure onset was at 2.5 yr of age. Events occurred during the awake state and consisted of staring followed by loss of tone ending with a short generalized tonicclonic seizure. Electroencephalography (EEG) recordings showed multifocal sharp waves. Her brain CT was normal. Initial unsuccessful treatment with valproic acid and clonazepam was followed by treatment with lamotrigine (25mg twice a day) in combination with valproic acid (200 mg TID). However, seizure frequency remained at one attack every 3 months.
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Six months after onset of epilepsy, her parents reported behavioral difficulties including aggressive behavior with temper tantrums, low frustration threshold, and hyperactivity that aggravated over time with the addition of involuntary laughter. Treatment with 0.5mg of risperidone was initiated to address these behavioral issues. The patient first visited our center at the age of 5 yr and presented as a non-dysmorphic normocephalic girl. Her neurological examination besides hyperactivity was within normal limits.
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Blood tests on admission showed hemoglobin A1c (HbA1c)-levels within the upper normal limit (6.4%; 46.64 mmol/mol). A thyroid function test taken while treated with valproic acid revealed a mild increase in TSH levels of 8.16mIU/L with normal free T4 levels (11 pmol/L). Cortisol and IGF-1 levels were within the normal range. The patient tested negative for antibodies to thyroglobulin, thyroid peroxidase, and endomysium, thereby excluding Hashimoto’s thyroiditis and celiac disease. The patient’s serum tested positive for IA2-Ab (80 U/mL), and negative for ZnT8-Ab. Insulin autoantibodies could not be determined because the patient was already being treated with exogenous insulin for 4 yr.
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CSF was obtained by lumbar puncture and revealed the presence of eight cells per mm3 (polynuclear and mononuclear) in presence of bloody tap. Flow cytometry analysis of these cells showed a normal profile for CD3+, CD4+, CD8+, CD2+, CD56+ and HLA-DR+ cells, and high frequencies of CD20+ B cells (577 CD20+ cells/mm3) (normal range: 50–300 CD20+ cells/mm3). Glucose and protein levels were within the normal range (126 and 47 mg/dL, respectively). Amino acids profiles and folates were within the normal range, as well as the 5 Methylene tetrahydrofolate reductase (5 MTHFR) level (84 nmol/L). Neurotransmitters profile demonstrated normal levels of 5-Hydroxytryptophan (5HTP), lDopa, 3-O-Methyldopa (3-OMD), 3-methoxy-4-hydroxyphenylglycol (MHPG), homovanillic acid (HVA)/5-Hydroxyindoleacetic acid (5-HIAA) and slightly elevated 5HIAA and HVA (407 and 1054 nmol/L, respectively). Neopterin level was within the normal range (13 nmol/L) while biopterin level was slightly elevated (43 nmol/L, while 30 nmol/L is the normal upper limit). Oligoclonal bands were not detected.
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Based on high GAD65Ab titers in both serum and CSF, the patient was treated with Vigabatrin, a GABA transaminase inhibitor to increase GABA concentrations in the brain and potentially decrease seizure frequency. While receiving 250 mg Vigabatrin BID for 4wk the patient had another seizure and the treatment was discontinued. Finally, a remission in the epileptic seizures was achieved after two courses of intravenous immunoglobulins [intravenous immunoglobulins (IVIg)] treatment (at a dosage of 2 g/kg body weight), and the parents reported an improvement in the patient’s behavior.
Methods The study was approved by the Human Investigations (Helsinki) Committee of Sheba Medical Center (#SMC-9275-12); written informed consent was obtained from the patient’s father.
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Determination of GAD65Ab titers Sera and CSF samples were analyzed using a radioligand binding assay as previously described (15). The cutoff for GAD65Ab positivity was 65 U/mL established as the 98th percentile of 50 healthy sera (standard curve’s range: 30–1000 U/mL). The sensitivity and specificity for T1D were 86 and 93%, respectively in the 2007 Diabetes Autoantibody Standardization Program (DASP) Workshop.
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Epitope-specific radioligand binding assay
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GAD65Ab epitope specificities were tested in a competitive epitope-specific radioligand binding assay (ES-RBA) as described (15). All sera and a GAD65Ab-positive control were analyzed for their binding to GAD65 in the presence of GAD65-specific recombinant Fab (rFab). rFab used in this study were derived from GAD65-specific human monoclonal antibodies. DPA and DPD were isolated from a patient with T1D and recognize epitopes located at amino acids 483–585 and 96–173, respectively (16). Monoclonal antibody DPD recognizes an epitope associated with mild beta cell autoimmunity (17), while DPA has not yet been associated with any specific disease. Monoclonal antibodies b96.11 and b78 were derived from a patient with autoimmune polyendocrine syndrome type 1 and recognize epitopes located at amino acid residues 308–365 and 451–585, respectively (18).
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Monoclonal antibody b96.11 shares its epitope specificity with the majority of T1D patients (15), while b78 is a prototypical epitope for GAD65Ab in SPS patients and only rarely bound by GAD65Ab present in T1D patients (9). The cutoff for specific competition was >15% as determined by control rFab D1.3 (15). Binding of GAD65Ab to GAD65 in the presence of rFab was expressed as: counts per minute (cpm) in the presence of rFab/cpm in the absence of rFab × 100. GAD65 enzyme activity assay GAD65 enzyme activity was measured by the 14CO2-trapping method described previously (19). The results are presented as: % residual activity=cpm in the presence of IgG/cpm in the absence of IgG × 100.
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Results GAD65Ab titer GAD65Ab titers of two independent serum samples collected from the patient at the ages of 5 and 6.5 yr, were very high (512 147 and 436 859 U/mL, respectively), compared with GAD65Ab titers ranging from 93 to 1865 U/mL in cohort of 10 well-characterized T1D children without neurological symptoms. Furthermore, the patient’s CSF sample had a GAD65Ab titer of 1113 U/mL, while CSF samples from two non-diabetic pediatric patients with epilepsy had GAD65Ab titers of 0 and 42 U/mL, respectively (Table 1). Inhibition of GAD65 enzyme activity
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The patient’s sera significantly reduced the GAD65 enzyme activity by 62 and 63%, respectively (Table 1). A control serum sample from an established SPS patient reduced the enzyme activity by 51%, consistent with previously reported inhibition of GAD65 enzyme activity by SPS samples (9, 11). None of the other samples, including the CSF sample of our patient, significantly reduced GAD65 enzyme activity. Epitope analysis Significant competition of binding of the above serum samples to GAD65 in the presence of rFab DPA, b96.11, b78, and DPD was observed (remaining binding was 78, 81, 61, and
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74%, respectively). A similar pattern was observed by the corresponding CSF sample, however, here competition with rFab DPD was significantly higher (46%) (Fig. 1). Significant competition of rFab for GAD65Ab in T1D patients was observed only for DPD (75%) and b96.11 (55%).
Discussion An association between T1D and epilepsy is suggested by the higher prevalence of epilepsy in patients with T1D (20, 21) and the increased prevalence of T1D in patients with epilepsy (22). Abnormalities of GABAergic function in animal models of epilepsy (23), epileptic seizures in GAD65-knock-out mice (24, 25), reduction of GABA levels in CSF and brain tissue of epileptic patients (26), and epileptic syndromes associated with GAD65Ab (21, 27) suggest that GAD65-specific autoimmunity may be linked to T1D-associated epilepsy.
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In this study we characterized the GAD65Ab of a young girl with T1D, followed by development of intractable epilepsy. Her EEG recordings showed multifocal sharp waves. Abnormal EEG changes, including abnormal slowing of background rhythms and epileptiform discharges, have been reported in newly diagnosedT1Dpatients presenting with Diabetic ketoacidosis (DKA) (28). However, in our patient epilepsy and EEG abnormalities were observed 1.5 yr after diagnosis with T1D and her diabetes was well controlled with no acute or recent episodes of DKA at the time of the EEG recording, therefore eliminating DKA as a contributing factor. Our patient’s GAD65Ab profile matched that of SPS patients in that her GAD65Ab were found both in the serum and the CSF, considerably exceeded titers found in T1D patients (7, 8), recognized the b78-defined epitope often bound by SPS patients (9), and most notably inhibited GAD65 enzyme activity. GAD65Ab in the CSF showed epitope specificities distinct from those present in the patient’s sera, consistent with intrathecalGAD65Ab production (12). Although the patient was diagnosed with T1D, her GAD65Ab only marginally recognized the b96.11-defined epitope associated with T1D (15). Previous studies found no differences in GAD65Ab epitopes associated with different neurological disorders (29), which is consistent with our finding of the SPS-associated GAD65Ab epitope in our patient. The finding that GAD65Ab in our patient inhibit GAD65 enzyme activity support the hypothesis of a pathologic role of these autoantibodies in epilepsy, similar as has been suggested for GAD65Ab in other neurological diseases (2, 3, 13). Different neurological symptoms may be due to distinct GAD65Ab distribution in the brain. IVIg treatment is often used to treat GAD65Ab-associated neurological disorders such as SPS, where treatment is accompanied by a decline of GAD65Ab titers (30).
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In this case, the patient showed some improvement of neurological symptoms after treatment with IVIg. Unfortunately, we were not able to obtain a sample within 2 months after IVIg treatment to test whether GAD65Ab titers decreased. It remains therefore unclear whetherGAD65Abhad a pathogenic relevance for the neurological symptoms in this patient, although clinically suggested by the reduction in seizure frequency and behavioral improvement.
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GAD65Ab may suppress synaptic GABA levels by two mechanisms: (i) reduction of GABA synthesis or (ii) interference with exocytosis of GABA (31, 32). The presence of the latter mechanism may explain why targeted inhibition of GABA transaminase with Vigabatrine did not induce measurable improvement of symptoms. Alternatively, inadequate dosage and short treatment time (4 wk) may have contributed to the unsuccessful outcome of this treatment. We have previously described an increased prevalence of SPS-like characteristics and neurological symptoms in a cohort of 100 T1D patients (8). In addition, we reported high GAD65Ab titers with SPS-like characteristics in a young girl with T1D, encephalitis, and epileptic seizures (33). Taken together with the current case, we suggest that GAD65Abassociated neurological disease may be under-diagnosed in pediatric T1D patients.
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Moreover high titers of GAD65Ab in patients with new onset T1D should alert the medical team of the possibility of an evolving neurological disease. We propose that careful titration and characterization of GAD65Ab, specifically assessment of enzyme inhibition and recognition of the b78-defined epitope, may be helpful in identifying T1D patients at risk for neurological diseases.
References
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30. Dalakas MC. The role of IVIg in the treatment of patients with stiff person syndrome and other neurological diseases associated with anti-GAD antibodies. J Neurol. 2005; 252(Suppl. 1):I19–I25. [PubMed: 15959668] 31. Vianello M, Tavolato B, Giometto B. Glutamic acid decarboxylase autoantibodies and neurological disorders. Neurol Sci. 2002; 23:145–151. [PubMed: 12536283] 32. Mitoma H, Song SY, Ishida K, Yamakuni T, Kobayashi T, Mizusawa H. Presynaptic impairment of cerebellar inhibitory synapses by an autoantibody to glutamate decarboxylase. J Neurol Sci. 2000; 175:40–44. [PubMed: 10785255] 33. Korff CM, Parvex P, Cimasoni L, et al. Encephalitis associated with glutamic acid decarboxylase autoantibodies in a child: a treatable condition? Arch Neurol. 2011; 68:1065–1068. [PubMed: 21825244] 34. Ludvigsson J, Heding L, Liedén G, Marner B, Lernmark A. Plasmapheresis in the initial treatment of insulin-dependent diabetes mellitus in children. Br Med J. 1983; 286:176–178. [PubMed: 6401518] 35. Hao W, Davis C, Hirsch IB, et al. Plasmapheresis and immunosuppression in stiff-man syndrome with type 1 diabetes: a 2-year study. J Neurol. 1999; 246:731–735. [PubMed: 10460456]
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Fig. 1.
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Epitope analysis. The capacity of the recombinant Fab (rFab) to inhibit GAD65 binding by human 65 kDa isoform of glutamate acid decarboxylase (GAD65Ab) was tested in a competitive epitope-specific radioligand binding assay (ES-RBA)(15). Samples [patient’s serum at 5 yr of age (P1), patient’s serum at 6.5 yr of age, 15 months following intravenous immunoglobulins (IVIG) treatment (P2), patient’s CSF (P CSF) and sera of 10 T1D patients (T1D)] were analyzed at a dilution representing half maximal antibody binding to GAD65. All samples were analyzed for their binding to GAD65 in the presence of rFab b96.11 (epitope amino acids 308–365), b78 (epitope amino acids 451–585),DPA(epitope amino acids 483–585), and DPD (epitope amino acids 96–173). Binding was expressed as cpm in the presence of rFab/cpm in the absence of rFab × 100. Mean binding with standard deviation of the means is shown. Horizontal bars indicate significant differences between two values.
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Table 1
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Inhibition of GAD65 enzyme activity GAD65Ab titer in U/mL
GAD65 enzyme activity (% residual activity)
Serum sample 1
512 147
38
Serum sample 2
436 859
37
CSF
1113
100
Control CSF 1
0
100
Control CSF 2
42
100
T1D (n=10)
93–1865
SPS
2435
100 49
CSF, cerebrospinal fluid; GAD65Ab, 65 kDa isoform of glutamate acid decarboxylase; SPS, stiff-person syndrome; T1D, type 1 diabetes.
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GAD65 enzyme activity presented as: % residual activity=cpm in the presence of IgG/cpm in the absence of IgG × 100. Serum sample 1, Serum sample 2 and ‘CSF’ were obtained form the study patient; Control CSF samples were from two non-diabetic pediatric patients with epilepsy; T1D – serum samples obtained from 10 newly diagnosed pediatric patients with type 1 diabetes (34), SPS – serum sample obtained from a patient with SPS (35).
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Pediatr Diabetes. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Pediatr Diabetes. 2016 December ; 17(8): 617–622. doi:10.1111/pedi.12346.
Epilepsy and behavioral changes, type 1 diabetes mellitus and a high titer of glutamic acid decarboxylase antibodies Esther Ganelin-Cohena,b, Dalit Modan-Mosesc,b, Rina Hemid, Hannah Kanetyd, Bruria Benzeeva,b,†, and Christiane S. Hampee,† aPediatric
Neurology Unit, The Edmond and Lily Safra Children’s Hospital, Chaim Sheba Medical Center, Ramat-Gan, Israel
Author Manuscript
bThe
Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel
cPediatric
Endocrinology and Diabetes Unit, The Edmond and Lily Safra Children’s Hospital, Chaim Sheba Medical Center, Ramat-Gan, Israel
dInstitute
of Endocrinology, Chaim Sheba Medical Center, Ramat-Gan, Israel
eDepartment
of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA, USA
Abstract
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Autoantibodies to the 65 kDa isoform of glutamate acid decarboxylase (GAD65Ab) are associated with a range of clinical disorders, including type 1 diabetes (T1D) and stiff-person syndrome (SPS). In this article we describe a young girl who was diagnosed with T1D at the end of her first year of life and developed drug-resistant epilepsy 18 months later, followed by behavioral disturbances. She was admitted to our center at the age of 5 yr, at which time high GAD65Ab titers were detected in the patient’s serum and cerebrospinal fluid (CSF). The titer remained elevated during 19 months of follow-up. Furthermore, GAD65Ab in both serum and CSF showed epitope binding characteristics similar to those observed for GAD65Ab in SPS patients, and GAD65Ab in the serum reduced GAD65 enzyme activity. Our results suggest an association between high GAD65Ab titers and epilepsy in children with T1D. Careful titration and characterization of GAD65Ab regarding inhibition of enzyme activity and epitope specificity may be helpful in identifying T1D patients at risk for neurological complications.
Author Manuscript
Keywords epilepsy; glutamate decarboxylase antibodies; immune-mediated; pediatric; type 1 diabetes Antibodies to the 65 kDa isoform of glutamate acid decarboxylase (GAD65Ab) are associated with several diseases, including type 1 diabetes (T1D) (80% of new onset patients), stiff-person syndrome (SPS) (60–80% of patients) (1), cerebellar ataxia (30–60%
Corresponding author: Esther Ganelin-Cohen, MD, PhD, Pediatric Neurology Unit, The Edmond and Lily Safra Children’s Hospital, Chaim Sheba Medical Center, Derech Sheba 2, Ramat-Gan, 52621, Israel. Tel: 972-35302687; 972-35302685; fax: 972-35305031; [email protected]. †Both authors contributed equally to this study.
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of patients) (2), intractable epilepsy (10–30% of patients) (for review see reference 3), and Batten disease (100% of patients) (4). Presence of GAD65Ab may predict development of neurological disorders as the antibodies may already be found in preclinical stages (5, 6).
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GAD65Ab in patients with neurological manifestation differ from those in T1D patients as manifested in titers exceeding those in T1D by at least 100-fold (7, 8). In addition, GAD65Ab from patients with neurological symptoms recognize both linear and conformational epitopes, while GAD65Ab from T1D patients without neurological complications only recognize conformational epitopes. (5), suggesting that the diseasespecific immune responses are reflected in GAD65Ab characteristics (9, 10). GAD65Ab in neurological diseases may have a pathological role mediated by antibody-induced inhibition of GAD65 enzyme activity (11). It is noteworthy that Gamma-Aminobutyric Acid (GABA) levels in the patients are often reduced (12). Furthermore, monoclonal GAD65Ab representing GAD65Ab specificities in neurological conditions lead to functional impairment of GABAergic synaptic transmission both in vitro and in vivo, while monoclonal GAD65Ab representing GAD65Ab specificities in T1D do not have these effects (13, 14). In this article we describe a case of a girl diagnosed with T1D, who presented with neurological manifestations and very highGAD65Ab titers. We characterized her GAD65Ab in serum and cerebrospinal fluid (CSF) regarding titer, epitope specificity, and inhibition of GAD65 enzyme activity.
Patient presentation Author Manuscript
A 5-yr-old girl with T1D was referred to our clinic for re-evaluation of intractable epilepsy. She was the youngest of five healthy siblings, born to healthy non-consanguineous Arab parents. The pregnancy was uneventful. During the first year of life she achieved normal developmental milestones. She was diagnosed with T1D at the age of 1 yr and was treated with insulin. Epileptic seizure onset was at 2.5 yr of age. Events occurred during the awake state and consisted of staring followed by loss of tone ending with a short generalized tonicclonic seizure. Electroencephalography (EEG) recordings showed multifocal sharp waves. Her brain CT was normal. Initial unsuccessful treatment with valproic acid and clonazepam was followed by treatment with lamotrigine (25mg twice a day) in combination with valproic acid (200 mg TID). However, seizure frequency remained at one attack every 3 months.
Author Manuscript
Six months after onset of epilepsy, her parents reported behavioral difficulties including aggressive behavior with temper tantrums, low frustration threshold, and hyperactivity that aggravated over time with the addition of involuntary laughter. Treatment with 0.5mg of risperidone was initiated to address these behavioral issues. The patient first visited our center at the age of 5 yr and presented as a non-dysmorphic normocephalic girl. Her neurological examination besides hyperactivity was within normal limits.
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Blood tests on admission showed hemoglobin A1c (HbA1c)-levels within the upper normal limit (6.4%; 46.64 mmol/mol). A thyroid function test taken while treated with valproic acid revealed a mild increase in TSH levels of 8.16mIU/L with normal free T4 levels (11 pmol/L). Cortisol and IGF-1 levels were within the normal range. The patient tested negative for antibodies to thyroglobulin, thyroid peroxidase, and endomysium, thereby excluding Hashimoto’s thyroiditis and celiac disease. The patient’s serum tested positive for IA2-Ab (80 U/mL), and negative for ZnT8-Ab. Insulin autoantibodies could not be determined because the patient was already being treated with exogenous insulin for 4 yr.
Author Manuscript
CSF was obtained by lumbar puncture and revealed the presence of eight cells per mm3 (polynuclear and mononuclear) in presence of bloody tap. Flow cytometry analysis of these cells showed a normal profile for CD3+, CD4+, CD8+, CD2+, CD56+ and HLA-DR+ cells, and high frequencies of CD20+ B cells (577 CD20+ cells/mm3) (normal range: 50–300 CD20+ cells/mm3). Glucose and protein levels were within the normal range (126 and 47 mg/dL, respectively). Amino acids profiles and folates were within the normal range, as well as the 5 Methylene tetrahydrofolate reductase (5 MTHFR) level (84 nmol/L). Neurotransmitters profile demonstrated normal levels of 5-Hydroxytryptophan (5HTP), lDopa, 3-O-Methyldopa (3-OMD), 3-methoxy-4-hydroxyphenylglycol (MHPG), homovanillic acid (HVA)/5-Hydroxyindoleacetic acid (5-HIAA) and slightly elevated 5HIAA and HVA (407 and 1054 nmol/L, respectively). Neopterin level was within the normal range (13 nmol/L) while biopterin level was slightly elevated (43 nmol/L, while 30 nmol/L is the normal upper limit). Oligoclonal bands were not detected.
Author Manuscript
Based on high GAD65Ab titers in both serum and CSF, the patient was treated with Vigabatrin, a GABA transaminase inhibitor to increase GABA concentrations in the brain and potentially decrease seizure frequency. While receiving 250 mg Vigabatrin BID for 4wk the patient had another seizure and the treatment was discontinued. Finally, a remission in the epileptic seizures was achieved after two courses of intravenous immunoglobulins [intravenous immunoglobulins (IVIg)] treatment (at a dosage of 2 g/kg body weight), and the parents reported an improvement in the patient’s behavior.
Methods The study was approved by the Human Investigations (Helsinki) Committee of Sheba Medical Center (#SMC-9275-12); written informed consent was obtained from the patient’s father.
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Determination of GAD65Ab titers Sera and CSF samples were analyzed using a radioligand binding assay as previously described (15). The cutoff for GAD65Ab positivity was 65 U/mL established as the 98th percentile of 50 healthy sera (standard curve’s range: 30–1000 U/mL). The sensitivity and specificity for T1D were 86 and 93%, respectively in the 2007 Diabetes Autoantibody Standardization Program (DASP) Workshop.
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Epitope-specific radioligand binding assay
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GAD65Ab epitope specificities were tested in a competitive epitope-specific radioligand binding assay (ES-RBA) as described (15). All sera and a GAD65Ab-positive control were analyzed for their binding to GAD65 in the presence of GAD65-specific recombinant Fab (rFab). rFab used in this study were derived from GAD65-specific human monoclonal antibodies. DPA and DPD were isolated from a patient with T1D and recognize epitopes located at amino acids 483–585 and 96–173, respectively (16). Monoclonal antibody DPD recognizes an epitope associated with mild beta cell autoimmunity (17), while DPA has not yet been associated with any specific disease. Monoclonal antibodies b96.11 and b78 were derived from a patient with autoimmune polyendocrine syndrome type 1 and recognize epitopes located at amino acid residues 308–365 and 451–585, respectively (18).
Author Manuscript
Monoclonal antibody b96.11 shares its epitope specificity with the majority of T1D patients (15), while b78 is a prototypical epitope for GAD65Ab in SPS patients and only rarely bound by GAD65Ab present in T1D patients (9). The cutoff for specific competition was >15% as determined by control rFab D1.3 (15). Binding of GAD65Ab to GAD65 in the presence of rFab was expressed as: counts per minute (cpm) in the presence of rFab/cpm in the absence of rFab × 100. GAD65 enzyme activity assay GAD65 enzyme activity was measured by the 14CO2-trapping method described previously (19). The results are presented as: % residual activity=cpm in the presence of IgG/cpm in the absence of IgG × 100.
Author Manuscript
Results GAD65Ab titer GAD65Ab titers of two independent serum samples collected from the patient at the ages of 5 and 6.5 yr, were very high (512 147 and 436 859 U/mL, respectively), compared with GAD65Ab titers ranging from 93 to 1865 U/mL in cohort of 10 well-characterized T1D children without neurological symptoms. Furthermore, the patient’s CSF sample had a GAD65Ab titer of 1113 U/mL, while CSF samples from two non-diabetic pediatric patients with epilepsy had GAD65Ab titers of 0 and 42 U/mL, respectively (Table 1). Inhibition of GAD65 enzyme activity
Author Manuscript
The patient’s sera significantly reduced the GAD65 enzyme activity by 62 and 63%, respectively (Table 1). A control serum sample from an established SPS patient reduced the enzyme activity by 51%, consistent with previously reported inhibition of GAD65 enzyme activity by SPS samples (9, 11). None of the other samples, including the CSF sample of our patient, significantly reduced GAD65 enzyme activity. Epitope analysis Significant competition of binding of the above serum samples to GAD65 in the presence of rFab DPA, b96.11, b78, and DPD was observed (remaining binding was 78, 81, 61, and
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74%, respectively). A similar pattern was observed by the corresponding CSF sample, however, here competition with rFab DPD was significantly higher (46%) (Fig. 1). Significant competition of rFab for GAD65Ab in T1D patients was observed only for DPD (75%) and b96.11 (55%).
Discussion An association between T1D and epilepsy is suggested by the higher prevalence of epilepsy in patients with T1D (20, 21) and the increased prevalence of T1D in patients with epilepsy (22). Abnormalities of GABAergic function in animal models of epilepsy (23), epileptic seizures in GAD65-knock-out mice (24, 25), reduction of GABA levels in CSF and brain tissue of epileptic patients (26), and epileptic syndromes associated with GAD65Ab (21, 27) suggest that GAD65-specific autoimmunity may be linked to T1D-associated epilepsy.
Author Manuscript Author Manuscript
In this study we characterized the GAD65Ab of a young girl with T1D, followed by development of intractable epilepsy. Her EEG recordings showed multifocal sharp waves. Abnormal EEG changes, including abnormal slowing of background rhythms and epileptiform discharges, have been reported in newly diagnosedT1Dpatients presenting with Diabetic ketoacidosis (DKA) (28). However, in our patient epilepsy and EEG abnormalities were observed 1.5 yr after diagnosis with T1D and her diabetes was well controlled with no acute or recent episodes of DKA at the time of the EEG recording, therefore eliminating DKA as a contributing factor. Our patient’s GAD65Ab profile matched that of SPS patients in that her GAD65Ab were found both in the serum and the CSF, considerably exceeded titers found in T1D patients (7, 8), recognized the b78-defined epitope often bound by SPS patients (9), and most notably inhibited GAD65 enzyme activity. GAD65Ab in the CSF showed epitope specificities distinct from those present in the patient’s sera, consistent with intrathecalGAD65Ab production (12). Although the patient was diagnosed with T1D, her GAD65Ab only marginally recognized the b96.11-defined epitope associated with T1D (15). Previous studies found no differences in GAD65Ab epitopes associated with different neurological disorders (29), which is consistent with our finding of the SPS-associated GAD65Ab epitope in our patient. The finding that GAD65Ab in our patient inhibit GAD65 enzyme activity support the hypothesis of a pathologic role of these autoantibodies in epilepsy, similar as has been suggested for GAD65Ab in other neurological diseases (2, 3, 13). Different neurological symptoms may be due to distinct GAD65Ab distribution in the brain. IVIg treatment is often used to treat GAD65Ab-associated neurological disorders such as SPS, where treatment is accompanied by a decline of GAD65Ab titers (30).
Author Manuscript
In this case, the patient showed some improvement of neurological symptoms after treatment with IVIg. Unfortunately, we were not able to obtain a sample within 2 months after IVIg treatment to test whether GAD65Ab titers decreased. It remains therefore unclear whetherGAD65Abhad a pathogenic relevance for the neurological symptoms in this patient, although clinically suggested by the reduction in seizure frequency and behavioral improvement.
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GAD65Ab may suppress synaptic GABA levels by two mechanisms: (i) reduction of GABA synthesis or (ii) interference with exocytosis of GABA (31, 32). The presence of the latter mechanism may explain why targeted inhibition of GABA transaminase with Vigabatrine did not induce measurable improvement of symptoms. Alternatively, inadequate dosage and short treatment time (4 wk) may have contributed to the unsuccessful outcome of this treatment. We have previously described an increased prevalence of SPS-like characteristics and neurological symptoms in a cohort of 100 T1D patients (8). In addition, we reported high GAD65Ab titers with SPS-like characteristics in a young girl with T1D, encephalitis, and epileptic seizures (33). Taken together with the current case, we suggest that GAD65Abassociated neurological disease may be under-diagnosed in pediatric T1D patients.
Author Manuscript
Moreover high titers of GAD65Ab in patients with new onset T1D should alert the medical team of the possibility of an evolving neurological disease. We propose that careful titration and characterization of GAD65Ab, specifically assessment of enzyme inhibition and recognition of the b78-defined epitope, may be helpful in identifying T1D patients at risk for neurological diseases.
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Fig. 1.
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Epitope analysis. The capacity of the recombinant Fab (rFab) to inhibit GAD65 binding by human 65 kDa isoform of glutamate acid decarboxylase (GAD65Ab) was tested in a competitive epitope-specific radioligand binding assay (ES-RBA)(15). Samples [patient’s serum at 5 yr of age (P1), patient’s serum at 6.5 yr of age, 15 months following intravenous immunoglobulins (IVIG) treatment (P2), patient’s CSF (P CSF) and sera of 10 T1D patients (T1D)] were analyzed at a dilution representing half maximal antibody binding to GAD65. All samples were analyzed for their binding to GAD65 in the presence of rFab b96.11 (epitope amino acids 308–365), b78 (epitope amino acids 451–585),DPA(epitope amino acids 483–585), and DPD (epitope amino acids 96–173). Binding was expressed as cpm in the presence of rFab/cpm in the absence of rFab × 100. Mean binding with standard deviation of the means is shown. Horizontal bars indicate significant differences between two values.
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Table 1
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Inhibition of GAD65 enzyme activity GAD65Ab titer in U/mL
GAD65 enzyme activity (% residual activity)
Serum sample 1
512 147
38
Serum sample 2
436 859
37
CSF
1113
100
Control CSF 1
0
100
Control CSF 2
42
100
T1D (n=10)
93–1865
SPS
2435
100 49
CSF, cerebrospinal fluid; GAD65Ab, 65 kDa isoform of glutamate acid decarboxylase; SPS, stiff-person syndrome; T1D, type 1 diabetes.
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GAD65 enzyme activity presented as: % residual activity=cpm in the presence of IgG/cpm in the absence of IgG × 100. Serum sample 1, Serum sample 2 and ‘CSF’ were obtained form the study patient; Control CSF samples were from two non-diabetic pediatric patients with epilepsy; T1D – serum samples obtained from 10 newly diagnosed pediatric patients with type 1 diabetes (34), SPS – serum sample obtained from a patient with SPS (35).
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