Clinical features and outcome of Wilson's disease with generalized epilepsy in Chinese patients

Abstract Objective Generalized epilepsy is rarely reported in patients with Wilson disease (WD) and lacks experience in clinical practice. We aim to provide better experience for the diagnosis and treatment for WD patients with epilepsy in the future. Methods A retrospective study was performed in 13 Chinese WD patients with generalized epilepsy. Each patient was diagnosed with WD by clinical evaluation and genetic screening. Patients were given small doses of antiepileptic drugs (AEDs), followed by copper‐chelation therapy when the seizures stabilized. Clinical manifestations, brain imaging changes, and treatment and outcome after a long‐term follow‐up were analyzed. Results Four out of 13 (30.8%) patients stopped taking copper‐chelation drugs for more than 1 year before they were admitted for epilepsy. The incidence of epilepsy of WD patients in our cohort is 1.43% (13/910), lower than those (4.5%‐5.9%) in other populations. After the attack of epilepsy, frontal lobes were the most common abnormalities (13/13, 100%) in patients, followed by brain stem (8/13, 61.5%) and thalamus (7/13, 53.8%). After a long‐term follow‐up, brain imaging and clinical manifestations of 8 (8/9, 88.9%) WD patients were significantly improved. Conclusions We firstly described WD patients with generalized epilepsy in the Chinese population. WD patients with aggravation of neuropsychiatric symptoms are prone to occur epilepsy; thus, brain MRI should be performed regularly in those patients. Cortical abnormality in brain MRI is a warning sign of epilepsy. Irregular use of copper‐chelation drugs and excessive copper deposition in the brain may be the cause of seizures. Long‐term standardized treatment for WD can effectively prevent the extensive brain damage and reduce the incidence of epilepsy in WD patients.


| INTRODUC TI ON
Wilson's disease (WD), one of the monogenic inherited diseases, is a kind of copper metabolic disturbance. The gene ATP7B mapping to chromosome13 (13q14.3) accounts for the disease, which encodes a P-type ATPase of transporting copper. 1 The deficiency of this protein prevents copper from being pumped out of the cell and therefore overloaded copper depositing in some specific organs, like liver, brain, cornea, and skeleton. 2 In brain, copper is more likely to accumulate in the basal ganglia, especially near the region of lenticular nucleus, followed by the brain stem. 3 The corresponding neuro-phenotypes related to the functional areas will appear, such as parkinsonism, tremor, and dystonia. The clinical heterogeneity and complications of WD increase the difficulties in diagnosis and treatment.
Although WD has common clinical manifestations, epilepsy and other secondary symptoms should not be ignored in the progress of the disease. In WD, abnormal deposition of copper or copper-induced damages could lead to brain function impairment and abnormal discharge of neurons, resulting in attacks of generalized epilepsy. 4,5 A rare event as it is, there is little experience in clinical practice. If the generalized epilepsy is not diagnosed and treated in time, the patients may progress into refractory epilepsy, missing the best opportunity for treatment, and their WD symptoms may deteriorate irreversibly. 6 During the last 15 years, we had diagnosed 910 unrelated WD patients by screening ATP7B mutations and found that 13 of them had attacks of generalized epilepsy. Here, we reviewed their clinical manifestations, brain imaging changes, and treatment and outcome, aiming to provide better experience for the diagnosis and treatment of WD patients with epilepsy in the future.

| Subjects
All patients were recruited from the Second Affiliated Hospital of Zhejiang University School of Medicine and Huashan Hospital of Fudan University. The study was approved by the ethics committees of these two hospitals, and the informed consents were obtained from the patients or their guardians. Among these 13 patients, the mutations carried by eight patients (P1-P8) and another 2 (P9-P10) had been reported, respectively, in our two previous studies. 7

| Mutation analysis
Extracting DNA from peripheral EDTA-treated blood was performed using Blood Genomic Extraction Kit (Qiagen) according to the manufactures' instructions. Sanger sequencing was performed on an ABI 3500xL Dx Genetic Analyzer (Applied Biosystems), and the procedure was described previously. 7,8 For patients who had only one variant detected by Sanger sequencing, we further performed multiplex ligation-dependent probe amplification assay (MLPA) using the ATP7B MLPA kit (SALSA P098-D1, MRC-Holland).

| Clinical characteristics of patients during the first visit
After analyzing 910 unrelated WD patients recruited between January 2004 and March 2019, we found that 13 (9 males and 4 females) of 910 (1.43%) patients were recorded to have generalized epilepsy. The pedigrees of them are shown in Figure 1A, and genetic analysis of 3 newly recruited patients (P11-P13) is shown in Figure 1B

| Clinical features of patients during hospitalization for generalized epilepsy
When generalized epilepsy occurred, the patient was admitted to the hospital. None of the patients had ascites. The clinical characteristics of these 13 patients during hospitalization for epilepsy are shown in Table 2. Assessment of liver function including Child-Pugh classification is shown in Table 3. Before epilepsy occurred, seven patients had a presentiment. Two patients (P2 and P11) presented spontaneous laughter or crying with no reason, and two (P7 and P10) complained of hallucinations. In addition, P3, P6, and P8 had formication, personality change, rant, and rave, respectively. During  Table 2. In addition to the basal ganglia, frontal lobes were the most common abnormalities (13/13, 100%), followed by brainstem (8/13, 61.5%) and thalamus (7/13, 53.8%). In the cortex, the rate of abnormalities in the parietal or temporal lobes was similar (3/13, 23.1%). In addition, P13, whose brain MRI showed extensive white matters, had some additional high signals in the cerebellum ( Figure 2A).
As a typical example, when he was 20 years old, P10 was initially diagnosed with WD due to difficulty in writing and speaking, and received copper-chelation treatment. He had status epilepticus at the age of 23 and was admitted to our center. According to his mother, he had been off his medication for about 7 months. He had hallucinations before the seizure. The frequency of seizures was about three times per day. In acute episode, the patient experienced intense contraction in his right limbs with eyes gazing to the left, following the alternating myoclonus, which lasted for nearly 1 minute. Then, the patient remained unconscious for nearly 20 minutes before fully waking up and suffered a long time of flaccid paralysis. We examined the patient and found that muscle strength was grade 0 in the right limbs and grade 4 in the left limbs, and the tendon reflexes of the extremities were reduced. During his hospitalization, several attacks of tonic seizures were also captured.

| Treatment and outcome
Once the diagnosis of WD is confirmed, copper-chelation treatment begins. Copper-chelation drugs used by patients before epilepsy are summarized in Table 4. Three patients (P2, P4, and P6) were treated with large doses of D-penicillamine (DPA) at their first visits. Four patients (P7, P9, P10, and P13) stopped taking F I G U R E 1 Pedigrees and mutation analysis. A, The pedigrees of 13 WD patients. Squares indicate males, circles indicate females, the black symbols indicate affected individuals, and arrows indicate the probands. B, Sequencing chromatograms and MLPA assay of three newly recruited patients (P11, P12, and P13). The upper panel depicts the reference sequence, and the lower panel represents the abnormal sequence copper-chelation drugs for more than 1 year before they were admitted to hospital for epilepsy. The rest of the patients followed the doctor's advice and had no adverse reactions. During the hospitalization for generalized epilepsy, copper-chelation drugs were suspended for a while and antiepileptic drugs (AEDs) were prescribed to ensure the effective control of seizures in a short time. As shown in Table 4, diazepam (DZP) was used to control status epilepticus and other AEDs like levetiracetam (LEV), carbamazepine (CBZ), and valproate (VPA) were applied to prevent the patients' seizures from recurring. No serious side effect was found in these patients. For example, AEDs (LEV 250 mg b.i.d. and VPA 500 mg q.d.) were given to P12 for months because of his multiple MRI abnormalities even though he had only one attack. When his condition was stable and no more seizures occurred within 1 month, the copper-chelation drugs were added to ensure the process of WD treatment.
In the long-term follow-up, two patients (P2, P6) died of irreversible neurological manifestations and poor physical conditions, two (P3, P7) were lost for some reasons, and the rest (P1, P4, P5, P8 to P13) were followed up at our center. There was no recurrence in 5 patients  Table 4). The clinical symptoms of P10 (1/9, 11.1%) did not improve due to the refusal of the drug, and the patient ended up bedridden.

| D ISCUSS I ON
Epilepsy occurred in WD patients are rare with only a few reports of secondary seizures in WD patients. 10 In the current study, we firstly reported Chinese WD patients with generalized epilepsy Compared to the generalized epilepsy rate (4.5%-5.9%) of WD patients previously reported, 6,11,12 the incidence of epilepsy (1.43%, 13/910) in our WD patients is lower. Our patients had multiple damages of organs, including brain, liver, and joint, and showed complex manifestations. The index of urinary 24-hour copper excretion at the time of initial diagnosis was at a high level, indicating a certain deposition of copper in the body. It is worth mentioning that 11 of 13 patients (84.6%) had psychiatric symptoms, such as irritability and unsociability, indicating extensive multiple injuries in the brain and prone to epilepsy. More attention and long-term follow-up should be given to these patients with severe neuropsychiatric phenotypes.
In this study, single AED or combination of two AEDs could control the seizures of most patients (12/13). The transition from two cases (P1 and P13) to partial epilepsy indicates that part of the damage is persistent, and AEDs should be used in a long term with small dose. Because patients had extensive brain damage, we do not recommend stopping AEDs in a short time. The evidence of drug withdrawal should be strictly in accordance with the guide. 13  T2-weighed FLAIR MRI is a preferred choice to find the associated lesions. 17 Abnormal copper deposition in lenticular nucleus is the most common in WD patients. 18 In a previous MRI study of WD, thalamus, midbrain, and pons are also regarded as common damaged areas. 19 In our patients, in addition to basal ganglia, the frontal lobe in cortical was also the most common damaged area (100%), followed by TA B L E 4 Treatment and outcome of 13 patients brain stem (61.5%), thalamus (53.8%), and parietal and temporal lobes (23.1%). Cerebellar injury is believed to occur in 4.4%-5.9% of WD patients 3,20 and occurred in one of our patients (7.7%). By comparison of brain MRI between WD patients before seizures, after seizures, and after treatment, the abnormalities caused by other diseases were excluded and some hidden lesions were found, which are helpful for the diagnosis. The increase or decrease of the signals in MRIs can reflect the course of the disease. Therefore, brain MRI can help to evaluate the progress of seizures and adjust the treatment in WD patients.
Although the brain MRIs have shown cortical and thalamic damages in patients with seizures, the origin of the seizures is difficult to determine and the mechanism underlying the extensive brain damage is not clear. There are many conjectures about the destruction of copper homeostasis in the body, such as copper overload, drug abuse, or quick discharge of copper. Abnormal neuron discharges in the cortex may be caused by copper changes or copper-mediated damages such as over oxidative stress, proinflammatory cytokines, and glutamate activation. 6 The cerebello-thalamo-cortical network has been proved to be able to explain some manifestations of Parkinson's disease and may be related to the cortical cell damage to a certain extent. 21 Excessive intake of copper in the early stage or discharge of too much copper at one time may lead to copper transfer into other areas. 22 In addition, copper-chelation drugs may be another main cause of the seizures in WD patients. It is reported that DPA may lead to elevated free copper concentration and enhanced oxidative stress in some parts of the body, resulting in irreversible neurological impairment. 23 Some first-generation antipsychotic drugs like chlorpromazine can also increase the risk of seizures, due to the neurotransmitter and neuropeptide alterations. 24,25

| CON CLUS IONS
In summary, we firstly described 13 WD patients with generalized epilepsy in the Chinese population. The incidence of epilepsy of WD patients (1.43%) is lower than those (4.5%-5.9%) of previous reports about other populations. Excessive copper deposition in the brain and irregular use of copper-chelation drugs may be the cause of epilepsy attacks. Long-term standardized treatment for WD can effectively prevent the extensive brain damage and reduce the incidence of epilepsy in WD patients.

ACK N OWLED G M ENTS
The authors sincerely appreciate the participants for their help and willingness to join in this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.