科技部補助專題研究計畫成果報告 期末報告
兒童癲癇的精準醫療:探討KCNQ2造成癲癇的致病機轉、基因型 與表現型的關係 與利用次世代定序探討台灣可行性與經濟性的
精準癲癇基因檢查
計 畫 類 別 ： 個別型計畫
計 畫 編 號 ： MOST 107-2314-B-040-021- 執 行 期 間 ： 107年08月01日至108年07月31日 執 行 單 位 ： 中山醫學大學醫學系兒童學科
計 畫 主 持 人 ： 李英齊
計畫參與人員： 碩士級-專任助理：黃瑞喜
中 華 民 國 108 年 11 月 18 日
中 文 摘 要 ： 背景: 癲癇佔人口約1%，而兒童至少有60%為找不到原因的癲癇 (epilepsy without an identified cause)。KCNQ2與KCNQ3為細胞 膜鉀離子通道調控基因。對帶有KCNQ2突變的人，會有癲癇、智障與 嚴重的腦病變，但目前尚未完全瞭解。
方法:本計劃目的主要在於探討癲癇病人的下列幾點(1) 探討不同 KCNQ2突變點基因型與表現型的關係，並進一步分析突變基因造成細 胞電流的改變，與病人症狀是否相關性。(3)對於過去對KCNQ2、
KCNQ3 突變陰性的病人，用次世代定序癲癇基因序列(epileptic panel)加做其他基因的檢查。(4)對國內的特有的突變點，將進一步 利用HEK293做功能性的分析。
結果: 在過去我們的初步研究結果，發現在64癲癇兒童中，做203個 基因序列，對病人的診斷率為13/65 (20%)，雖然與選擇的病人有關
，但是跟國外其他的報告比較相似，約20-30%。這顯示癲癇基因序 列(epileptic panel)的診斷率仍不足。帶有病理性基因突變方式包 括missense mutations、與non-sense mutations。我們發現
KCNQ2致病的突變點包括: p.S247L、c.387+1 C>A(splicing)、
p.V543M、p.R432C、p.P285T、p.T287I、p.R581X、p.T287I與 p.R553W。這些分別造成輕度到非常嚴重的腦病變。
結論: KCNQ2 致病的突變佔兒童不明原因癲癇(epilepsy without an identified cause)5%。但在第一次抽筋小於 2個月的新生兒佔 13%。對於過去對KCNQ2與KCNQ3突變陰性的病人我們用次世代定序串 聯序列(epileptic genetic panel)203基因。發現在64癲癇兒童中
，對病人的診斷率為20%，雖然與選擇的病人有關，但是跟國外其他 的報告比較相似。這顯示癲癇基因序列的診斷率仍不足。可利用其 他方法來共用或取代來檢測癲癇基因的成因。
中 文 關 鍵 詞 ： KCNQ2；癲癇；功能性研究; 癲癇基因序列
英 文 摘 要 ： Background and Objectives: Epilepsy without an identified cause in newborns and children is usually genetic
heterogeneously. Molecular diagnosis is valuable for the patient and family. Genetic findings influence management and specific precision therapies are emerging. Numerous genes encode for epilepsy. One of them is KCNQ2. KCNQ2 mutations can contribute to benign familial neonatal convulsions (BFNC), benign familial neonatal-infantile seizures (BFNIS), benign familial infantile seizures (BFIS), and neonatal-onset epileptic encephalopathy (EE).
Despite some case-series reports and some functional studies, however, the phenotypes and genotypes are still complex and noteworthy.
Patients and Methods: The KCNQ2 sequencings done were selected from 131 nonconsanguineous pediatric epileptic patients (age range: 2 days to 18 years) with non-lesional epilepsy. We will use epileptic panel including 203 genes to study the KCNQ2 negative epileptic infants and children.
To investigate amount of the membranous KCNQ2 protein expression and its relationship to disease by cytoplasmic
and membranous protein separation Those KCNQ2 mutation variants were transfected into HEK293 cells to investigate functional changes for all mutations. The functional study includes whole cell double-patch-clamp and cytoplasmic and membranous KCNQ2 protein expression to investigate the electrical changes in cell membranes.
Results: Seven (5%) index patients had verified KCNQ2 mutations: c.387+1 G>T (splicing), c.1741 C>T (p.Arg581*), c.740 C>T p.(Ser247Leu), c.853 C>A p.(Pro285Thr), c.860 C>T p.(Thr287Ile), c.1294 C>T p.(Arg432Cys), and c.1627 G>A p.(Val543Met). We found, after their paternity had been confirmed, that three patients had de novo p.(Ser247Leu), p.(Pro285Thr), and p.(Thr287Ile) mutations and neonatal- onset epileptic encephalopathy; however, their frequent seizures remitted after they turned 6 months old. Those with the c.387+1 G>T (splicing), (p.Arg581*), and
p.(Val543Met) mutations presented with benign familial neonatal convulsions. In addition to their relatives, 14 patients had documented KCNQ2 mutations, and 12 (86%) had neonatal seizures. The seizures of all 5 patients treated with oxcarbazepine remitted.
Membrane protein expression in all variants was
significantly (p < 0.05) lower than in the wild-type. The most severe variants in the phenotype were R213Q, S247L and S247W then E515D, V543M and S247X. The protein level was lowest in E515D and highest in T857I. E515D more
significantly (p < 0.05) lowered protein expression than did T857I, correlated with phenotype severities. R213Q, however, protein expression on HEK cell membranes more than did other variants except T857I but was not correlated with phenotype severities.
Conclusion: KCNQ2-related epilepsy led to varied outcomes (from benign to severe) in our patients. KCNQ2 mutations accounted for 13% of patients with seizure onset before 2 months old in our study. KCNQ2 mutations can cause
different phenotypes in children. The p.(Pro 285Thr) is a novel mutation, and the p.(Pro 285Thr), p.(Ser247Leu), and p.(Thr287Ile) variants can cause neonatal-onset epileptic encephalopathy. Our findings support the notion that KCNQ2 variants contribute to surface protein expression change are not necessarily correlated with the phenotype.
英 文 關 鍵 詞 ： KCNQ2; epilepsy; functional study; epileptic panel
KCNQ2 mutations in childhood non-lesional epilepsy: Variable phenotypes and a novel
1mutation in a case series
2Inn-Chi Lee1,2*, Tung-Ming Chang3, Jao-Shwann Liang4, Shuan-Yow Li2,5 3
1Division of Pediatric Neurology, Department of Pediatrics, Chung Shan Medical University 4
Hospital, Taichung, Taiwan.
5
2Institute of Medicine, School of Medicine, Chung Shan Medical University, Taichung, 6
Taiwan.
7
3Department of Biological Science and Technology, College of Biological Science and 8
Technology, National Chiao Tung University, Hsinchu, Taiwan.
9
4Department of Pediatrics, Far Eastern Memorial Hospital, New Taipei City, Taiwan.
10
5Genetics Laboratory and Department of Biomedical Sciences, Chung Shan Medical 11
University, Taichung, Taiwan.
12 13
Number of words (abstract): 216; Number of words (main text): 2989; Number of tables: 4 14
(main 2; supplementary 2); Number of figures:7 (main 5; supplementary 2); Number of color 15
figures: 6 (figures 1, 3, 4, 5; supplementary 1 and 2) 16
Supplementary materials: 4 (2 supplementary tables and 2 supplementary figures) 17
Running title: KCNQ2-associated childhood epilepsy
18*
Reprint requests and correspondence to:
19
Inn-Chi Lee, MD, PhD 20
Institute of Medicine, School of Medicine, Chung Shan Medical University, Taichung, 21
Taiwan. #110, Section 1, Chien-Kuo North Road, Taichung 402, Taiwan 22
Tel: +886-4-2473-9535; Fax: +886-4-2471-0934; E-mail: [email protected]
2324 25
1
ABSTRACT
26Background: Epilepsy caused by a KCNQ2 gene mutation usually manifests as neonatal
27seizures during the first week of life. The genotypes and phenotypes of KCNQ2 mutations are 28
noteworthy.
29
Methods: The KCNQ2 sequencings done were selected from 131 nonconsanguineous
30pediatric epileptic patients (age range: 2 days to 18 years) with non-lesional epilepsy.
31
Results: Seven (5%) index patients had verified KCNQ2 mutations: c.387+1 G>T (splicing),
32c.1741 C>T (p.Arg581
*
), c.740 C>T p.(Ser247Leu), c.853 C>A p.(Pro285Thr), c.860 C>T 33p.(Thr287Ile), c.1294 C>T p.(Arg432Cys), and c.1627 G>A p.(Val543Met). We found, after 34
their paternity had been confirmed, that three patients had de novo p.(Ser247Leu), 35
p.(Pro285Thr), and p.(Thr287Ile) mutations and neonatal-onset epileptic encephalopathy;
36
however, their frequent seizures remitted after they turned 6 months old. Those with the 37
c.387+1 G>T (splicing), (p.Arg581
*
), and p.(Val543Met) mutations presented with benign 38familial neonatal convulsions. In addition to their relatives, 14 patients had documented 39
KCNQ2 mutations, and 12 (86%) had neonatal seizures. The seizures of all 5 patients treated
40with oxcarbazepine remitted.
41
Conclusion: KCNQ2-related epilepsy led to varied outcomes (from benign to severe) in our
42patients.KCNQ2 mutations accounted for 13% of patients with seizure onset before 2 months 43
old in our study. KCNQ2 mutations can cause different phenotypes in children. p.(Pro 285Thr) 44
is a novel mutation, and the p.(Pro 285Thr), p.(Ser247Leu), and p.(Thr287Ile) variants can 45
cause neonatal-onset epileptic encephalopathy.
46
Keywords: KCNQ2, childhood epilepsy, phenotypes, epileptic encephalopathy
4748 49 50
2
1 INTRODUCTION
51KCNQ2 (OMIM 602235) mutations can contribute to benign familial neonatal
52convulsions (BFNC) (Biervert et al., 1998; Leppert et al., 1989; Neubauer et al., 2008), 53
benign familial neonatal-infantile seizures (BFNIS), benign familial infantile seizures (BFIS), 54
and neonatal-onset epileptic encephalopathy (EE) (Kato et al., 2013; Weckhuysen et al., 2013, 55
2012). The mutant gene, KCNQ2, a voltage-gated potassium-channel gene at 20q13, is 56
usually inherited with autosomal-dominant form in a benign epileptic syndrome associated 57
with a KCNQ2 mutation. Patients with BFNC, BFNIS, or BFIS usually have seizures as 58
neonates and infants, but they are predicted to have benign outcomes (Leppert et al., 1989;
59
Neubauer et al., 2008; Singh et al., 1998).Most seizures will spontaneously disappear during 60
an infant’s first 12 months of life (Coppola et al., 2003; Singh et al., 1998). In KCNQ2 61
mutation-associated neonatal-onset EE, most mutations are de novo or mosaic-inherited, and 62
patients present with severe seizures and severe neurological outcomes (Kato et al., 2013;
63
Weckhuysen et al., 2012). Electroencephalograms (EEGs) in neonatal-onset EE patients show 64
interictal burst-suppression or multiple focal spikes (Kato et al., 2013; Weckhuysen et al., 65
2012). Patients usually have intellectual developmental delays despite seizure remission. A 66
loss (Maljevic et al., 2011; Maljevic, Wuttke, & Lerche, 2008; Wuttke et al., 2008) or gain 67
(Miceli et al., 2015; Millichap et al., 2017) of KCNQ2 gene function is presumed to be the 68
major mechanism for KCNQ2-associated neonatal-onset EE. Recent analyses of data from 69
genome-wide association studies (GWASs) of humans and animals indicate that KCNQ2 70
mutations contribute to schizophrenia susceptibility (Choi et al., 2018; Lee, Kim, & Song, 71
2013). However, outcomes for patients with KCNQ2 mutations cannot be accurately 72
predicted.
73
The KCNQ2 gene is expressed predominantly in the brain and encodes for voltage-gated 74
potassium-channel subunits that underlie the M-current, a repolarizing current that limits 75
3
repetitive firing during long-lasting depolarizing inputs (Wang et al., 1998; Cooper, 76
Harrington, Jan, & Jan, 2001; Cooper & Jan, 2003; Coppola et al., 2003). Each subunit of 77
KCNQ2 consists of heteromultimeric channels with six transmembrane domains (S1-S6):
78
voltage sensors in S1-S4, a loop between S5 and S6 that builds the ion channel pore domain, 79
and a long C-terminal region of mostly unknown function (Biervert et al., 1998; Cooper et 80
al., 2001; Lerche et al., 1999). The C-terminal tail contains two helical domains (A and B) 81
that bind to calmodulin (CaM), a calcium (Ca2+) sensor (Ambrosino et al., 2015). Helix A 82
contains the consensus CaM binding IQ motif, and helix B mediates Ca2+-dependent CaM 83
binding (Ambrosino et al., 2015; Liu & Devaux, 2014; Zhou et al., 2016). CaM accounts for 84
trafficking protein to cell-surface membranes. The mutations in the CaM domain have been 85
reported to impair the interaction with calmodulin molecules, and to impair surface 86
expression of potassium channel, which increased action potential firin
g and hyperexcitability
87(Maljevic et al., 2008; Zhou et al., 2016).
88
The precise percentage of neonates and children with KCNQ2-associated epilepsy is 89
unknown. About 163 (1.9%) of the 8565 patients in one study (Lindy et al., 2018) with 90
epilepsy and neurodevelopmental disorders had detectable KCNQ2 mutations.Weckhuysen et 91
al. (2013) reported 11 (13%) KCNQ2-associated neonatal-onset seizures in 84 patients with 92
neonatal-onset EE. Kato et al. (2013)identified 12 (5%) KCNQ2-associated cases of 93
neonatal-onset EE in 239 patients. The percentages of KCNQ2-associated epilepsy were not 94
consistent but depended upon what kinds of patients were enrolled. For neonates, rapidly 95
diagnosing and promptly stopping seizures should improve the patient’s outcome (Chen et al., 96
2018; Grinton et al., 2015).The diagnosis can be supported by clinical features, EEG 97
findings, age seizure onset, and family history, and it can be confirmed using a genetic study.
98
Despite some case-series reports (Grinton et al., 2015; Kato et al., 2013; Weckhuysen et 99
al., 2013, 2012) and some functional studies (Maljevic et al., 2011, 2008; Wuttke et al., 100
4
2008), however, the phenotypes and genotypes are still complex and noteworthy. We 101
previously reported (Lee, Yang, Liang, Chang, & Li, 2017) in a functional study of HEK293 102
cells that genotype is a major determinant of phenotype. Additional investigations are 103
warranted. In the present study, we investigated a series of cases with KCNQ2 mutation 104
variants from patients with childhood non-lesional epilepsy.
105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
5
2 MATERIALS AND METHODS
1272.1 Ethical Compliance
128Ethical approval of the study was provided by Chung Shan Medical University Hospital’s 129
Internal Review Board (IRB #: CS13036).
130
2.2 Recruiting participants
131One hundred thirty-one patients met all three criteria for “childhood epilepsy without an 132
identified cause” ([1] first seizure when < 18 years old, [2] age at last visit < 18 years old, and 133
[3] at least one magnetic resonance image (MRI) with no detectable seizure-related lesions) 134
and were enrolled in the study. Seizure onset occurred before 2 months old for 45 (34%) 135
patients, and between 2 months and 18 years old for the other 86 (66%). The KCNQ2 genes 136
were sequenced and screened in all patients. If mutations were detected, we requested to 137
sequence and screen the patients’ relatives. Fifty-five healthy adults who said that they had 138
never had epileptic seizures were enrolled as controls.
139
2.3 Extracting and amplifying DNA from KCNQ2 exons using a polymerase chain
140reaction
141A genomic DNA purification kit (Gentra Systems; http://www.gentra.com) was used to 142
extract a genomic DNA sample from a peripheral whole blood sample from each patient after 143
we obtained informed consents. All 17 exons of the KCNQ2 gene were amplified using a 144
polymerase chain reaction (PCR) for each patient.
145
2.4 Purifying and sequencing PCR products
146The PCR products were then purified (PCR-M Clean-Up System; Viogene-Biotek Corp., 147
New Taipei City, Taiwan), and their concentrations were measured using a spectrophotometer 148
(Ultrospec 3100 Pro; Amersham Biosciences UK, Little Chalfont, Buckinghamshire, UK).
149
The products were sequenced using an automated DNA sequencer (3100; Applied 150
Biosystems, Foster City, CA). DNA sequencing was done using a kit (ABI PRISM BigDye 151
6
Terminator Cycle Sequencing Ready Reaction Kit, v3.1; Applied Biosystems) on the ABI 152
PRISM 3730XL DNA analyzer. The sequence data of each patient were checked against the 153
GenBank reference sequence and version number of KCNQ2 gene (NM_172107.3). Each 154
mutation was numbered and described based on the Mutation Database Initiative 155
(MDI)/Human Genome Variation Society (HGVS) Mutation Nomenclature 156
Recommendations (<http://www.hgvs.org/mutnomen> or 157
<http://www.HGVS.org/varnomen>).
158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
7
3 RESULTS
187Seven (5%) of the 131 patients (3 boys; 4 girls) had KCNQ2 mutations: one each for 188
c.1627 G>A c.1627 G>A p.(Val543Met); c.1294 C>T p.(Arg432Cys); c.740 C>T 189
p.(Ser247Leu); c.1741 C>T (p.Arg581
*
); c.853 C>A p.(Pro285Thr); c.860 C>T 190p.(Thr287Ile); and a splicing mutation: c.387+1 G>T (Tables 1 and 2). None of these 7 191
mutations was found in the control group (Supplemental Table 1). All 7 mutations, except one 192
splicing and one nonsense mutation, were missense. Four parents had the same mutations as 193
did their children, but three children had de novo mutations: p.(Ser247Leu), p.(Pro285Thr), 194
and p.(Thr287Ile). Mutation p.(Ser247Leu) was in the S5 transmembrane domain; mutations 195
p.(Pro285Thr) and p.(Thr287Ile) were in the pore domain (Figure 1); the mutation in patient 196
3 was in a splicing site in the S2 domain (Figure 1); and mutation p.(Val543Met) was in the 197
C-terminal CaM domain (Figure 1).
198
The three patients with the de novo p.(Ser247Leu), p.(Pro285Thr), and p.(Thr287Ile) 199
mutations had neonatal EE (Table 1). Three index patients had BFNC. One index patient with 200
p.(Arg432Cys) had rolandic spikes in EEGs when awakening and prominent spikes when 201
sleeping, which is consistent with continuous spikes and waves during slow-wave sleep 202
(CSWS) (Table 1). The pedigrees of the 7 patients’ families are shown in Figure 2.
203
For patients whose first seizure onset occurred before they were 2 months old, the 204
positive rate for a KCNQ2 mutation was 13% (6/45), and the negative rate was 87% (39/45).
205
This was significantly (p < 0.001) different from that of patients whose first seizure onset 206
occurred after they were 2 months old: their negative rate for a KCNQ2 mutation was 99%.
207
3.1 Patients with sporadic KCNQ2 mutations had neonatal-onset EE and severe
208neurodevelopmental outcomes
2093.1.1 Patient 4 had the de novo p.(Ser247Leu) mutation in the S5 domain.
210
Patient 4 had the c.740 C>T p.(Ser247Leu) mutation (Figure 3), frequent neonatal 211
8
seizures, and apnea. His EEG shows multiple focal spikes (Figure 3). He was treated with 212
multiple antiseizure drugs: phenobarbital, vigabatrin, and clonazepam. After he had turned 4 213
months old, he was treated with oxcarbazepine (OXC) and his seizures abated. An MRI 214
showed basal ganglion hyperdensity (Figure 3). He also had a severe cognitive disability and 215
could not walk at 3 years old.
216
3.1.2 Patients 6 and 7 had the de novo p.(Pro285Thr) and p.(Thr287Ile) mutations in the
217pore domain.
218
Patient 6 had the de novo p.(Pro285Thr) mutation, frequent neonatal seizures, and apnea.
219
Her EEG (Figure 4) showed burst-suppression. Her amplitude-integrated EEG (aEEG) 220
monitor showed many seizures (Figure 5: arrows) with unique low-voltage fast activity 221
arising from the left hemisphere and followed by rhythmic theta and delta rhythms and 222
postictal extremely low voltage activity during ictal recordings. She had neonatal seizures 223
and apnea. The seizures became less frequent after she turned 2 months old, but she had a 224
severe cognitive disability. The patient was treated with multiple antiseizure drugs:
225
phenobarbital, phenytoin, vigabatrin, and clonazepam. The seizures abated 2 months after she 226
had been treated with OXC.
227
Patient 7 had the de novo p.(Thr287Ile) (Supplementary Figure 1: lower) and presented 228
with neonatal seizures. OXC and topiramate controlled his seizures. He could walk, but he 229
had a severe cognitive disability at 3 years old.
230
3.2 Patients with familial KCNQ2 mutations had relatively benign neurodevelopmental
231outcomes
2323.2.1 Patient 2 had the c.1294 C>T p.(Arg432Cys) mutation
233The computer-based SIFT and PolyPhen algorithms predicted that the p.(Arg432Cys) 234
mutation was deleterious. The Arginine (R) at protein position 432 is highly conserved in 235
mammals. The patient had a mild epileptic phenotype; her first seizure (febrile) was at 1 year 236
9
old, and she had an afebrile seizure at 5 years old. She was then treated with OXC, had two 237
more seizures, and was referred to the hospital. After topiramate (2 mg/kg/day) had been 238
added, her seizures remitted. She had mild cognitive impairment. A genetic study showed that 239
both she and her father, who had seizures as an infant, had the p.(Arg432Cys) mutation. An 240
extensive epileptic panel study that included 203 genes found no other genetic defect 241
responsible for her seizures (Supplementary Table 2). Her EEG showed bilateral central 242
spikes during awakening and prominent spikes after sleeping. The spike waves clearly 243
activated during sleep, compared with their EEG tracings while awake. The spike-wave index 244
is over 50% in non-REM sleep and there were fewer sleep spindles visible, which is 245
consistent with CSWS (Supplementary Figure 2) (Lesca et al., 2012; Scheltens-de Boer, 246
2009). Her MRI was unremarkable, and her seizures remitted after she turned 7 years old.
247
3.2.2 Patient 1 had the c.1627 G>A p.(Val543Met) mutation
248This case was previously reported (Lee, Yang, Liang, Chang, & Li, 2017). The patient’s 249
first seizure occurred when she was 2 weeks old. She had asymmetrical general tonic seizures.
250
Her most recent previous seizure had been when she was 2 months old. Her seizures remitted 251
after she had begun taking OXC when she was 2 months old. Her MRI was unremarkable.
252
The family history showed that the patient’s father and two of her aunts also had the 253
p.(Val543Met) mutation and had had seizures from birth, but they had no cognitive 254
disabilities. At 6 years and 2 months old, her cognitive development was normal.
255
3.2.3 Patient 3 had the c.387+1 G>T (splicing) mutation
256Patient 3’s first seizure occurred when he was 3 days old. The seizures were general 257
tonic-clonic seizures with lip cyanosis. He was first treated with intravenous phenobarbital, 258
after which the seizures temporarily remitted. At 1 year old, the patient had another general 259
tonic-clonic seizure and was treated with OXC. His MRI and neurodevelopment were 260
unremarkable. The family history showed that the patient’s mother had had seizures from 261
10
birth. A genetic study for KCNQ2 showed that both he and his mother had the c.387+1 G>T 262
mutation but no KCNQ3 mutation (Supplementary Figure 1: upper).
263
3.2.4 Patient 5 had the c.1741 C>T (p.Arg581 * ) mutation
264Patient 5’s first seizure was a general tonic-clonic seizure with lip cyanosis when she 265
was 3 days old. The seizures remitted after she had been treated with oral phenobarbital. Her 266
MRI and neurodevelopment were unremarkable at 4 years old. The family history showed 267
that the patient’s mother and sister had had seizures from birth. A genetic study for KCNQ2 268
showed that all three had the c.1741 C>T(p.Arg581
*
) mutation.269
3.3 Age at onset of seizures, and relapse of seizures after 3 years old
270Seizure onset occurred in 6 of the 7 index patients when they were younger than 1 271
month and in 1 index patient when she was older than 1 month (febrile seizures at 1 year old).
272
In the 7 families, we found 14 patients with confirmed KCNQ2 mutations. Seizure onset 273
occurred at younger than 1 month in 12 (86%) and at older than 1 month in 2 (14%) (1 with 274
infantile seizures [1 month to 1 year old]; 1 with febrile seizures at 1 year old) 275
We confirmed KCNQ2 mutations in 14 patients, 6 (43%) of whose seizures continued 276
after they were older than 3 years. All 7 index patients had general tonic or clonic seizures.
277
EEGs showed burst-suppression or multiple focal spikes in 3 index patients, focal discharges 278
in 3, and CSWS in 1.
279
3.4 Drug treatment
280One of the 7 index patients was treated with only one antiseizure drug (patient 1: OXC);
281
6 were treated with more than 1 (4 were treated with OXC, and their seizures remitted).
282
Seizures in all 7 patients completely or partially remitted after 6 months of drug treatments 283
(Table 1).
284 285 286
11
4 DISCUSSION
287Our most important finding is that KCNQ2 mutations led to a variety of phenotypes in 288
childhood epilepsy, e.g., neonatal-onset EE, BFNC, and CSWS. We found KCNQ2 mutations 289
in about 5% of non-lesional childhood epilepsy patients, and in about 13% of patients with 290
neonatal seizure onset when they were younger than 2 months. We found three de novo 291
mutations (p.(Ser247Leu), p.(Pro285Thr), and p.(Thr287Ile)), all of which are in critical 292
KCNQ2 domains. p.(Pro285Thr) is a novel mutation. Patients with one of these de novo
293mutations had worse outcomes.
294
The p.(Ser247Leu) and p.(Ser247Trp) mutations are pathogenic 295
(https://www.ncbi.nlm.nih.gov/clinvar), as is (p.Ser247*) (Dedek, Fusco, Teloy, & Steinlein, 296
2003). Interestingly, each leads to a different phenotype. The p.(Ser247Leu) and 297
p.(Ser247Trp) cause neonatal-onset EE; however, (p.Ser247*) causes benign neonatal 298
convulsions(Hunter et al., 2006).Kato et al. (2013) reported a de novo mutation p.(Pro285His) 299
that causes Ohtahara syndrome. The global allele frequencies of p.(Ser247Leu), 300
p.(Pro285Thr), and p.(Thr287Ile) are zero, according to the ExAC browser 301
(http://exac.broadinstitute.org/). In our case, p.(Pro285Thr) was de novo, novel, and highly 302
likely to cause neonatal-onset EE, according to the guidelines of the American College of 303
Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG) 304
(Richards et al., 2015). The p.(Thr287Ile) was classified as a variant of uncertain significance 305
(VOUS) (https://www.ncbi.nlm.nih.gov/clinvar). In patient 7, who had the p.(Thr287Ile) 306
variant, p.(Thr 287Asn) was also pathogenic (Milh et al., 2013), which supports the finding 307
that p.(Thr287Ile) is pathogenic. We hypothesize that p.(Ser247Leu), p.(Pro285Thr), and 308
p.(Thr287Ile) contribute to neonatal-onset EE, and that all three should be classified as 309
pathogenic.
310
We also found that KCNQ2-associated epilepsy patients had varied outcomes. Two 311
12
regions involved with the important KCNQ2 functional domains—S1-S6 and CaM—might 312
cause neonatal-onset BFNC. However, in the most benign cases, the neurodevelopmental 313
outcomes were relatively better. If the epilepsy is hereditary, seizure remission usually occurs 314
after the patient turns 3 years old, and most such patients undergo normal cognitive 315
development (Claes et al., 2004; Grinton et al., 2015; Singh et al., 2003).
316
We also found c.1545 G>C p.(Glu515Asp) in 5 patients with varied phenotypes: BFNC, 317
CSWS, and unclassified epilepsy syndrome. However, they were excluded from the case 318
series because of conflicting interpretations of p.(Glu515Asp)’s pathogenicity, despite our 319
report (Lee, Yang, & Li, 2017)that there was a functional current change in HEK293 cells 320
transfected with p.(Glu515Asp). Patients with p.(Glu515Asp) were associated with a mild 321
BFNC phenotype, but in some patients, it was associated with cognitive delay and 322
attention-deficit hyperactive disorder (ADHD). Dravet syndrome patients with the p.(Glu515 323
Asp) mutation more often have developmental delays than do Dravet syndrome patients 324
without p.(Glu515Asp) (Hammer et al., 2017). It is probable that other mutations of modified 325
genes contributed to the phenotype. The global allele frequency of p.(Glu515Asp) is 326
0.002499, according to the ExAC browser (http://exac.broadinstitute.org/). The Exac 327
population includes patients with Tourette’s syndrome and schizophrenia. Amino acid 328
changes from E (glutamic acid) to D (aspartic acid) can cause a thermophilic change of 329
protein. The p.(Glu515Asp) is in the CaM domain, which means that its functional position is 330
relatively important. However, to determine the pathogenicity of the p.(Glu515Asp) mutation, 331
additional accurate age-matched case-control studies are necessary. We also found three other 332
benign KCNQ2 variants: c.2264A>G p.(Tyr755Cys), c.1253G>T p.(Gly418Val), and 333
c.51G>C p.(Glu17Asp). The p.(Glu17Asp) variant is novel, and after a segregation study, we 334
hypothesized that it was benign.
335
Although BFNC are considered benign, BFNC patients might have cluster seizures, 336
13
which inevitably require drug control to prevent secondary brain injury (Grinton et al., 2015).
337
In neonatal EE patients, OXC, valproic acid, topiramate, vigabatrin, and clonazepam were 338
used to treat seizures (Kato et al., 2013; Weckhuysen et al., 2013, 2012). OXC was 339
considered more efficacious for KCNQ2-associated seizures in several studies (Grinton et al., 340
2015; Pisano et al., 2015; Sands et al., 2016), which is consistent with our findings. However, 341
responses to antiseizure drugs require additional investigation.
342 343
5 CONCLUSIONS
344KCNQ2 mutations accounted for 5% of all non-lesional pediatric epilepsy and 13% of
345patients with seizure onset before 2 months old. KCNQ2 mutations can cause variable 346
phenotypes in children, from BFNC to severe neonatal-onset EE. The p.(Ser247Leu), p.(Pro 347
285Thr), and p.(Thr287Ile) mutations can cause neonatal-onset EE.
348 349
Acknowledgments
350We thank Yi-Ho Weng and I-Ting Chen for performing the electroencephalography 351
(EEG) with great patience. We thank Dr Pi-Chuan Fan from National Taiwan University 352
Hospital for his valuable assistance. This work was supported by grant 107-2314-B-040-021 353
from the Taiwan Ministry of Science and Technology.
354
Conflict of Interest
355The authors declared that they have no conflicts of interest.
356 357 358 359 360 361
14
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490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
20
Table 1. Seven of the 131 patients had identified KCNQ2mutatoins. The clinical and familial 512
histories are summarized.
513
NA, not available; VOUS, variance of unknown significance; PHT, phenytoin; OXC, oxcarbazepine; VPA,
514
valproic acid; TOP, topiramate; PB, phenobarbital; KEP, levetiracetam; SAB, vigabatrin; CLN, clonazepam;
515
MRI, magnetic resonance imaging; EEG, electroencephalography; +++, daily; ++, weekly; +, less than weekly;
516
ADHD, attention deficit and hyperactivity; Dev. Del./Int. Dis., Developmental delay/intellectual disability. The
517
sequence data of each patient were checked against the GenBank reference sequence and version number of
518
KCNQ2 gene (NM_172107.3).
519
Patient number Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Genotype c.1627 G>A c.1294 C>T c.387+1 G>T c.740 C>T c.1741 C>T c.853 C>A c.860 C>T
Protein change p.(Val543Met) p.(Arg432Cys) Splicing p.(Ser247Leu) (p.Arg581*) p.(Pro285Thr) p.(Thr287Ile) Family mutation Father and two
aunts
Father Mother De novo Mother
and sister
De novo De novo
Sex Female Female Male Male Female Female Male
Other genetic study
KCNQ3 Panel KCNQ3 Whole exon Panel Whole exon KCNQ3
Family number of seizures other than index patient (n)
3 1 1 0 2 0 0
Later seizures older than 3 years from familial KCNQ2 mutation (n)
0 2 0 1 1 1 1
Age at first seizure
Day 14 1year (febrile seizure)
Day 3 Day 3 Day 3 Day 2 Newborn
Seizure type General tonic General tonic General clonic
General tonic General tonic General tonic General tonic
Seizure
frequency before drug control
+ + +++ +++ +++ +++ +++
Drug control OXC OXC, TOP PB, OXC Intravenous PB, PHT then changed to PB, SAB, CLN
PB Intravenous PB, PHT then changed to oral PB, SAB, CLN, OXC
TOP, OXC
Seizure
frequency after 6-months drugs
- - - - - - +
Abnormal MRI No No No Basal ganglion No Thin corpus
callosum
No
Dev. Del./Int. Dis. No Mild(ADHD) No Severe No Severe Severe
21
Table 2. Genotypes and phenotypes in 7 KCNQ2 mutations.
Genotype (N) Phenotype Severity NCBI ClinVar Functional Global East Asia Cont . FATHMM Poly SIFT ACMG
domain MAF
MAF
in Taiwan
(N=55)
predict Phen2 score
c.387+1 G>T Splicing BFNC + Pathogenic S2 0 0 0 PVS1, PM2, PP4
c.740C>T p.(Ser247Leu) missense EE +++ Pathogenic S5 0 0 0 D D D PS2, PM1, PM2,PP3, PP4
c.853C>A p.(Pro285Thr) missense EE +++ Novel Pore domain 0 0 0 D D D PS2, PM1,PM2, PP3, PP4, PM5
c.860C>T p.(Thr287Ile) missense EE +++ VOUS Pore domain 0 0 0 D D D PS2, PM1, PM2, PP4, PP3
c.1294C>T p.(Arg432Cys) missense CSWS + VOUS C-terminal 4.53E-05 0 0 D D D PM2, PP3
c.1627G>A p.(Val543Met) missense BFNC + VOUS Calmodulin 0 0 0 D D D PS3, PM2, PP3, PP4
c.1741C>T (p.Arg581*) nonsense BFNC + Pathogenic C-terminal 0 0 0 PVS1, PM2, PP4
BFNC indicates benign familial neonatal convulsions; EE, neonatal-onset epileptic encephalopathy; CSWS, continuous spikes and waves during slow-wave sleep;
NCBI ClinVar: National Center for Biotechnology Information, clinical variability and predictability (https://www.ncbi.nlm.nih.gov/clinvar); Cont, Control of healthy adults without seizures; Global MAF: global mutation allele frequency in EXAC browser; East Asia MAF
: East Asia mutation allele frequency in EXAC browser;
VOUS, variant of uncertain significance; FATHMM predict: Functional Analysis Through Hidden Markov Models prediction; D, damage; T, tolerant. ACMG, American College of Medical Genetics and Genomics and the Association for Molecular Pathology. The sequence data of each patient were checked against the GenBank reference sequence and version number of KCNQ2 gene (NM_172107.3).
22
KCNQ2-associated childhood epilepsy
FIGURE LEGENDS
Figure 1. Seven mutation variants associated with KCNQ2 functional domains. CaM:
calmodulin domain.
Figure 2. The pedigrees in 7 index families are shown.
Figure 3. Sanger sequencing shows that patient 4 has a de novo c.740C>T p.(Ser247Leu)
mutation (right lower, arrow), and an interictal EEG shows multiple focal spikes (left upper).The EEG improved after the patient turned 6 months old (left lower). His MRI shows hyperdensities (right upper, arrow) in his bilateral basal ganglia. He had frequent (> 10 per day) neonatal seizures.
Figure 4. Patient 6 has a de novo c.853C>A p.(Pro285Thr) mutation (left lower) and an EEG
that shows neonatal epileptic encephalopathy with burst-suppression (left upper). Her EEG improved and her seizures attenuated after 2 months. The MRI shows a thin corpus callosum (right upper, arrows, and right lower).Figure 5. Patient 6’s amplitude-integrated EEG monitor showed many seizures (arrows) with
unique low-voltage fast activity arising from the left hemisphere followed by rhythmic theta and delta rhythms and postictal extremely low voltage activity during ictal recordings.SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Patient 3 has a KCNQ2 splicing mutation: c.387+1G>T (upper,
arrow). Seizure onset occurred on postnatal day 3. Her mother had the same mutation. The phenotype in the patient and her mother was BFNC. Patient 7, with the c.860 C>Tp.(Thr287Ile) mutation (lower, arrow), presented with a neonatal burst-suppression EEG and a seizure.
Supplementary Figure 2. Patient 2 had her first febrile seizure at 1 year old and an afebrile
seizure at 5 years old. Her intellectual development was mildly abnormal. A genetic study showed that she and her father had the c.1294 C>T p.(Arg432Cys) mutation (right lower,23
KCNQ2-associated childhood epilepsy
arrow) and seizures. The computer-based SIFT/PolyPhen algorithms predicted that the p.(Arg432Cys) mutation was deleterious. Her EEG shows bilateral central spikes when awakening (left upper) and marked accentuated spikes after she falls asleep, which is consistent with CSWS (right upper). Her MRI is unremarkable (left lower).
24
1
Severe developmental delay/intellectual disability Normal intelligence or
mild developmental delay/intellectual disability
CaM
CaM
p.(Val543Met)
p.(Arg432Cys) c.387+1G>T p.(Ser247Leu)
(p.Arg581*)
p.(Pro285Thr) p.(Thr287Ile)
Patient 1 Patient 2 Patient 3
Patient 4 Patient 5 Patient 6
Patient 7
Normal intelligence
Severe developmental delay/intellectual disability Mild developmental delay/intellectual disa bility
Wild type -/-
1
1
Supplementary Table 1. Genotypes from 55 adults without a history of seizures.
Chr,chromosome; het, SNP,
single-nucleotide polymorphism; heterozygous; ref, reference; var, variance; fre, frequency; AA Var, amino acid
variance; N/A, non available. The sequence data of each patient were checked against the GenBank reference sequence and version number ofKCNQ2 gene (NM_172107.3).
Chr Position Type Zygosity Genotype Ref Var
Var Freq (%)
dbSNP AA Var
Econ 17 c.2619+248 C > T chr20 62037749 SNP Het G/A G A 41.8 rs6122440
Exon 17 c.2238 T > A chr20 62038378 SNP Het A/T A T 14.5 rs1801471 p.(Pro746=) Intron 16 c.1888-29G > A chr20 62038757 SNP Het C/T C T 18.2 rs3746364
Exon 15 c.1719C > T chr20 62044847 SNP Het G/A G A 3.6 N/A p.(Ala573=) Intron 13 c.1525+57C > T chr20 62046199 SNP Het G/A G A 9.1 N/A
Intron 13 c.1525+55G > A chr20 62046201 SNP Het C/T C T 7.3 N/A Intron 13 c.1525+53T > C chr20 62046203 SNP Het A/G A G 12.7 N/A
Exon 6 c.912C > T chr20 62070966 SNP Het G/A G A 40.0 rs2297385 p.(Phe304=) Intron 2 c.388-26G > T chr20 62076743 SNP Het C/A C A 6.7 rs6062939
Intron 1 c.296+112T > A chr20 62103409 SNP Het A/T A T 27.3 N/A
Exon 1 c.127G > A chr20 62103690 SNP Het C/T C T 25.4 N/A p.(Ala43Thr)
Supplementary Table 2. The epileptic panel included 203 genes for patient 2.
AARS (16q22.1) CLN6 (15q21-q23) HCN4 (15q24-q25) NRXN1 (2p16.3) SLC12A5 (20q13.12)
ABAT (16p13.3) CLN8 (8p23) HDAC2 (6q21) NTNG1 (1p13.3) SLC13A5 (17p13.1)
ABCD1 (Xq28) CNTNAP2 (7q35-q36) HDAC4 (2q37.3) OPHN1 (Xq12) SLC1A1 (9p24.2) ABCB1 (7q21.1) CPA6 (8q13.2) HNRNPU (1q44) OPRM1 (6q24-q25) SLC25A12 (2q31.1) ADGRV1(5q14.3) CSNK1G1 (15q22.31) HUWE1 (Xp11.22) PAFAH1B1 (17p13.3) SLC25A19 (17q25.3) ADSL (22q13.1) CSTB (21q22.3) IER3IP1 (18q21.1) PCDH19 (Xq22) SLC25A22 (11p15.5)
ALG13 (Xq23) CTSD (11p15.5) ITPA (20p13) PCNT (21q22.3) SLC35A2 (Xp11.23)
ALDH7A1 (5q31) DCX (Xq22.3-q23) KCNA1 (12p13) PDHA1 (Xp22.12) SLC6A1 (3P25.3) ARFGEF2 (20q13.13) DEPDC5 (22q12.2-q12.3) KCNA2 (1p13.3) PHF6 (Xq26.3) SLC2A1 (1p35-p31.3) ARHGEF9 (Xq22.1) DNM1(9q34.11) KCNB1 (20q13.13) PIGA (Xp22.2) SLC9A6 (Xq26.3) ARHGEF15(17p13.1) DOCK7 (1p31.3) KCNAB1 (3q26.1) PIGO (9p13.3) SNIP1 (1p34.3) ARX (Xp22.13) EFHC1 (6p12-p11) KCNC1 (11p15.1) PLCB1 (20p12) SMS (11p11.2) ARV1(1q42.2) EEF1A2 (20q13.33) KCNH5 (14q23.2) PNKP (19q13.4) SPTAN1 (9q33-q34) ASAH1(8p22) EFHC2 (Xp11.3) KCNJ10 (1q23.2) PNPO (17q21.32) SRPX2 (Xq21.33-q23)
ASPM (1q31) EMX2 (10q26.1) KCNJ11 (11p15.1) POLG (15q25) STIL (1p33)
ATP1A2 (1q23.2) EPM2A (6q24) KCNMA1 (10q22.3) PPT1 (1p32) ST3GAL3 (1p34.1)
ATP1A3 (19q13.2) FASN (17q25.3) KCNQ2 (20q13.3) PRICKLE1 (12q12) ST3GAL5 (2p11.2)
ATP6AP2 (Xp11.4) FLNA (Xq28) KCNQ3 (8q24) PRICKLE2 (3p14) STRADA (17q23.3)
ATP7A (Xq21.1) FLVCR2 (14q24.3) KCNT1 (9q34.3) PRRT2 (16p11.2) STXBP1 (9q34.1) AMT (3p21.2-p21.1) FOLR1 (11q13.3-q13.5) KCNV2 (9p24.2) PTCH1 (9q22.3) STX1A (7q11.23) ATR (3q22-q24) FOXG1 (14q13) KCTD7 (7q11.21) PTEN (10q23.31) STX1B (16p11.2) BRD2 (6p21.3) FOXH1 (8q24.3) KPNA7 (7q22.1) RS1 (Xp22.13) SYN1 (Xp11.4-p11.2)
CACNA1A (19p13) FRRS1L (9q31.3) LBR (1q42.1) RELN (7q22) SYNJ1 (21q22.11)
CACNA1H (16p13.3) GABRA1 (5q34-q35) LGI1 (10q23.33) SCARB2 (4p21.1) SYNGAP1 (6p21.32) CACNB4 (2q22-q23) GABRB3 (15q11.2-q12) LPCAT1 (5p15.33) SCN10A (3p24.2-p22) SZT2 (1p34.2) CASK (Xp11.4) GABRD (1p36.3) MAGI2 (7q21) SCN11A (3p24-p21) TBC1D24 (16p13.3) CASR (3q13.3-q21) GABRG2 (5q31.1-q33.1) MBD5 (2q23.1) SCN1A (2q24) TCF4 (18q21.2) CCL2 (17q11.2-q12) GAMT (19p13.3) MCPH1 (8p23) SCN1B (19q13.1) TGIF1 (18p11.3) CDK5RAP2 (9q33.3) GATM (15q15.3) ME2 (18q21) SCN2A (2p24.3) TPP1 (11p15.5)
CDKL5 (Xp22) GFAP (17q21.31) MECP2 (Xq28) SCN2B (11q23) TRPM2 (21q22.3)
CDON (11q24.2) GNAO1 (16q13) MEF2C (5q14) SCN3A (2q24) TSEN2 (3p25.1)
CENPJ (13q12.2) GLDC (9p24.1) MFSD8(4q28.1-q28.2) SCN3B (11q23.3) TSEN34 (19q13.4) CEP152 (15q21.1) GLI2 (2q14) MTHFR (1p36.3) SCN4A (17q23.1-q25.3) TSEN54 (17q25.1) CHD2 (15q26.1) GOSR2 (17q21.32) NCAM1 (11q23.2) SCN4B (11q23) UBE3A (15q11-q13)
CHRNA2 (8p21) GUF1 (4p12) NECAP1 (12p13.31) SCN5A (3p21) VANGL1 (1p13)
CHRNA4 (20q13.2-q13.3) GPR56 (16q13) NDE1 (16p13.1) SCN7A (2q21-q23) WDR62 (19q13.12)
CHRNB2 (1q21) GRIN1 (9q34.3) NDUFA1 (Xq24) SCN8A (12q13)
WWOX (16q23.1-23.2)
CLCN2 (3q26) GRIN2A (16p13) NEDD4L (18q21.31) SCN9A (2q24) ZEB2 (2q22)
CLCN4 (Xp22.2) GRIN2B (12p13.1) NF1 (17q11.2) SHH (7q36) ZIC2 (13q32)
CLN3 (16p12.1) HCN1 (5p12) (5p12) NHLRC1 (6p22.3) SIK1 (21q22.3) CLN5 (13q21.1-q32) HCN3 (1q22) NODAL (10q22.1) SIX3 (2p21)
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107年度專題研究計畫成果彙整表
計畫主持人：李英齊 計畫編號：107-2314-B-040-021-
計畫名稱：兒童癲癇的精準醫療:探討KCNQ2造成癲癇的致病機轉、基因型與表現型的關係 與利用次 世代定序探討台灣可行性與經濟性的精準癲癇基因檢查
成果項目 量化 單位
質化
（說明：各成果項目請附佐證資料或細 項說明，如期刊名稱、年份、卷期、起 訖頁數、證號...等）
國 內
學術性論文
期刊論文 0
研討會論文 0 篇
專書 0 本
專書論文 0 章
技術報告 0 篇
其他 0 篇
智慧財產權 及成果
專利權 發明專利 申請中 0
件
已獲得 0
新型/設計專利 0
商標權 0
營業秘密 0
積體電路電路布局權 0
著作權 0
品種權 0
其他 0
技術移轉 件數 0 件
收入 0 千元
國
外 學術性論文 期刊論文 4 篇
1.Inn-Chi Lee, Tung-Ming Chang, Jao-Shwann Liang, Shuan-Yow Li.
KCNQ2 mutations in childhood non- lesional epilepsy: variable
phenotypes and a novel mutation in a case series. Molecular genetics and genomic medicine.
2019;7(7):e00816. doi:
10.1002/mgg3.816
2. Inn-Chi Lee, Jiaznn-Jou Yang, Shuan-Yow Li. KCNQ2-Associated Epilepsy: A Review of Variable Phenotypes and Neurodevelopmental Outcomes. Neuropsychiatry.
2018;8(1):318–323.
3.Lin CH, Lin WD, Chou IC, Inn-Chi Lee, Hong SY Epilepsy and
Neurodevelopmental Outcomes in Children With Etiologically
Diagnosed Central Nervous System Infections: A Retrospective Cohort Study. Front Neurol. 2019;10:528.
doi: 10.3389/fneur.2019.00528.
4.Epileptic spasms in PPP1CB- associated Noonan-like syndrome: A case report with clinical and therapeutic implications. Chien- Heng Lin, Wei-De Lin, I-Ching Chou, Inn-Chi Lee, Hueng-Chuen Fan,
Syuan-Yu Hong. BMC Neurology. 2018;
20;18(1):150.
研討會論文 3
1.Inn-Chi Lee, Jao-Shwann Liang, Ming-yuh Chang, Tung-Ming Chang, Pi-Chuan Fan, Jiann-Jou Yang. Ictal and interictal
electroencephalographic findings in KCNQ2-associated childhood
epilepsy. AOCCN, 2019, Malaysia.
2.Inn-Chi Lee, Jiaznn-Jou Yang, Shuan-Yow Li. Medical Exome Sequencing (MES) in Childhood Severe Epilepsy after Negative Candidate Genes Analysis. 2019, 33rd International Epilepsy Congress. Bangkok, Thailand.
3.Inn-Chi Lee, Jiaznn-Jou Yang, Shuan-Yow KCNQ2 Mutations in Childhood Non-lesional Epilepsy:
Variable Phenotypes and novel
mutations in a Cases Series. Infant seizure society, 2018, Roman,
Italy.
專書 0 本
專書論文 0 章
技術報告 0 篇
其他 0 篇
智慧財產權 及成果
專利權 發明專利 申請中 0
件
已獲得 0
新型/設計專利 0
商標權 0
營業秘密 0
積體電路電路布局權 0
著作權 0
品種權 0
其他 0
技術移轉 件數 0 件
收入 0 千元
參 與 計 畫 人 力
本國籍
大專生 0
人次
碩士生 0
博士生 0
博士級研究人員 0
專任人員 0
非本國籍
大專生 0
碩士生 1 11 個月碩士專任助理
博士生 0
博士級研究人員 0
專任人員 0
其他成果
（無法以量化表達之成果如辦理學術活動
、獲得獎項、重要國際合作、研究成果國 際影響力及其他協助產業技術發展之具體 效益事項等，請以文字敘述填列。）
1.每年均邀請Baylor Medical School, USA, Lee Jun C.
Wong (Professor, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA)舉辦研討會並合作分子診斷工具
2. 協助生技基因公司探討分子診斷工具與分析結果
