Case Report: Extraocular muscles paralysis associated with GAD65 antibody: a case series study

Objective To explore the clinical manifestations of glutamic acid decarboxylase 65 (GAD65) antibody-positive patients with extraocular symptoms and the possible mechanism. Method Assays for the presence of GAD65 antibodies were performed on patients’ serum and cerebral spinal fluid (CSF). The brain and ocular structures involved in eye movement were assessed via magnetic resonance imaging (MRI). Tests such as electromyography (EMG), particularly repetitive nerve stimulation (RNS), and neostigmine tests were utilized for differential diagnosis. Additionally, the interaction of GAD65 antibodies with muscle tissue was confirmed using immunofluorescence techniques. Result Each patient exhibited symptoms akin to extraocular myasthenia gravis (MG), with two individuals reporting diplopia and two experiencing ptosis. GAD65 antibodies were detected in either the serum or CSF, which were shown to bind with monkey cerebellum slides and mouse muscle slides. Neuroimaging of the brain and extraocular muscles via MRI showed no abnormalities, and all patients tested negative for the neostigmine test, RNS via EMG, and the presence of MG antibodies. However, thyroid-related antibodies were found to be abnormal in four of the patients. Conclusion Our results showed that GAD65 antibodies are not only associated with encephalitis, cerebellum ataxia or stiff-person syndrome caused by the decrease of GABAergic transmission but also diplopia and ptosis. Therefore, we should pay more attention to extraocular muscle paralysis patients without pathogenic antibodies directed against the components of neuromuscular junctions.


Introduction
Glutamic acid decarboxylase (GAD) is an enzyme that catalyzes the conversion of the inhibitory neurotransmitter g-aminobutyric acid (GABA) to glutamate.It is selectively expressed in nerve terminals of presynaptic GABAergic neurons and pancreatic b cells (1).Previous studies reported that Anti-GAD65 antibodies (GAD65-Abs) could be seen in patients with progressive cerebellar ataxia, limbic encephalitis, epilepsy, myelitis, palatal tremor, myoclonus, and even a helpful biomarker of stiff-person syndrome (SPS) (2,3).
Most of the previous studies have focused on the role of GAD65-Abs interfering in GABAergic synaptic transmission, and some researchers assumed that the inflammatory cascade induced by GAD65-Abs is the cause of neuronal loss and cerebellar atrophy.But there are few studies on its possible role in muscle and neuromuscular junction.Moreover, only 1 case has reported GAD65-Abs and abnormal eye movement (4).We found that some patients with ptosis or diplopia accompanied by thyroidassociated antibodies positive were misdiagnosed as MG and ineffective treatment.
The present study analyzed extraocular muscle activity, antibody titers, and immunofluorescence assays of antibody b i n d i n g t o m u s c l e m e m b r a n e s i n f o u r G A D 6 5 -A b s positive patients.

Study population
Patients with diagnosed ptosis and eye movement irregularities who tested positive for GAD65-Abs in either serum or CSF were selected for this study.These patients were admitted to either The First Affiliated Hospital of Sun Yat-sen University or The Second Affiliated Hospital of Guangzhou Medical University from January 1, 2022, to December 31, 2022.Prior to inclusion in this research, every patient gave their informed written consent, and the study secured ethical approval.

Anti-GAD65 antibody assay
Stored at -80°C, serum and CSF samples were assessed for GAD65 antibody presence.The analysis was performed utilizing a commercial kit (Euroimmun, Luebeck, Germany) designed to assay anti-GAD65 antibody, which employs a GAD65-transfected cell line derived from human embryonic kidney 293 cells, as delineated previously (5).

Immunofluorescence assay for GAD65 antibody
A tissue-based assay confirmed the positive samples of CSF and serum.Briefly, serum (1:10) or CSF(1:50) diluted in phosphatebuffered saline (PBS) was reacted with monkey cerebellum tissue slides provide by IbnSinaHealth (Guangzhou) Technology Co.,Ltd and mouse muscle slides for 3 h at room temperature.The slides were rinsed twice with PBS and then incubated with fluoresceinconjugated goat anti-human IgG for 2 h.Finally, the slides were rinsed with PBS, and the fluorescence intensity was examined under a microscope (6).

Results
During the study period, 14 patients were detected GAD65-Abs positive in serum or CSF.Four of these patients with extraocular symptoms were identified and included in the study.The patient's neurological symptoms and examination results are summarized in the table.All complained of diplopia, and physical examination showed limited eye movement, vertical or horizontal, without misalignment, and no abnormal pupil diameter or light reflection was found.(Figure 1).All patients had an abnormality of thyroidrelated antibodies; two (patients 1 and 3) had ptosis; All patients without other symptoms and signs of myopathy.The neostigmine test, RNS of EMG and the antibodies of MG including anti-AchR, anti-Musk and anti-LRP4 were negative in all patients.In order to distinguish brainstem encephalitis and cranial nerve injury associated with GAD65-Abs, brain and orbit MRIs were performed in all patients with diplopia.
Specifically, patient 1 presented with ptosis and diplopia, with no significant variations observed between morning and evening.MRI scans of the brain, cranial nerve and extraocular muscles were normal (Figure 2).Despite negative results for MG-related antibodies and EMG, an initial misdiagnosis of MG was made, but treatment with pyridostigmine bromide, glucocorticoid therapy, and intravenous human immunoglobulin demonstrated poor efficacy.Patient 2 experienced isolated diplopia following a night shift and had a history of leukoderma, which required melanin grafting two years prior.CT and MRI scans conducted for this patient showed no abnormalities.Patient 3 presented with unilateral right-sided ptosis in 2017, with no abnormalities in antibodies of MG, neostigmine test, RNS, brain and orbital MRI.No efficacy of pyridostigmine bromide treatment were observe.But the patient experienced symptom relief within a few months without recurrence even stopped taking medicine.She developed epilepsy in November 2022.Subsequent MRI scans of the brain suggested bilateral hippocampal and adjacent gyrus swelling, suggestive of autoimmune encephalitis.Patient 4 exhibited diplopia with minor ocular discomfort.In addition, she experienced dryness of the eyes and mouth, but sicca syndrome indices were negative, and an MRI scan appeared normal.Interestingly, patients 1, 3, and 4 displayed poor responses to high-dose methylprednisolone treatment but showed marked improvement following therapy with plasma exchange (PE) or immunoadsorption.
To explore the possible interaction of GAD65-Abs and diplopia, we incubated the serum and CSF with monkey brain tissue and mouse muscle slides.We found that serum and CSF IgG bound to neurons in the granular layer of the monkey cerebellum, consistent with GAD65 distribution characteristics (Figure 3).Furthermore,  This study has several limitations.Primarily, the small sample size and the specific focus on the dysfunction of eye muscles without a broader investigation of the involvement of other skeletal muscles.We observed that patients with GAD65 antibody positive in current case have isolated clinical characteristics of extraocular muscle dysfunction, suggesting that in clinical diagnosis and treatment, attention should be paid to identifying whether there is abnormal expression of GAD65 in patients with unknown extraocular muscle dysfunction.However, it is not clear whether GAD65 antibody plays a major role in the pathogenic mechanism of these patients, and it is still necessary to further study and clarify the relevant mechanism of interaction between GAD65 antibody and cranial nerve or extraocular muscle.Our findings serve as a springboard for future research into the cellular and molecular mechanisms of GAD65-Abs in neurological syndromes.

1
FIGURE 1 Extraocular paralysis of a typical patient.(A1) Right side ptosis at stage of first onset.(A2) Bilateral ptosis at stage of second onset.(A3) Bilateral eyelids after treatment.(B) Eye movement at stage of onset.(C) Eye movement after treatment.

FIGURE 2
FIGURE 2 The brain and orbital MRI are normal.(A-C) The T1-weighted, T2 FLAIR and T1-weighted contrast-enhanced sequence of barin.(D-F) The T1weighted, T2 FLAIR and T1-weighted contrast-enhanced sequence showed no ocular muscle abnormalities.(G) The orbital coronal MRI is normal.(H, I) The T2 FLAIR and T1-weighted contrast-enhanced sequence of orbital sagittal MRI.(J) The CTA of cranial arteries.(K, L) The MRI of oculomotor nerve in T2 Space and T1-weighted contrast-enhanced sequence is normal.