Common genetic variants that alter the risk of developing epilepsy

1. Common genetic
variants that alter the
risk of developing
epilepsy
Larry Baum

2. People
• Patient samples
– Virginia Wong (Paediatrics, HKU)
– Ping Wing Ng (United Christian Hosp.)
– Colin Lui (Tseung Kwan O Hospital)
– Ngai Chuen Sin (HA Head Office)
– Tak Hong Tsoi (PYN Eastern Hospital)
• Control samples
– Annie Kung (Medicine, HKU)
– Neil Thomas (Health, U. Birmingham)
– Brian Tomlinson (Medicine, CUHK)
– Yu Lung Lau (Paediatrics, HKU)
– Wanling Yang (Paediatrics, HKU)
• Cells, expression, & genotyping
– Shuai Zhang (Pharmacy, CUHK)
– Chunbo Zhang (Pharmacy, CUHK)
• Data analysis and bioinformatics
– Clara Tang (Psychiatry, HKU)
– Johnny Kwan (Psychiatry, HKU)
– Ben Yip (Psychiatry, HKU)
– Su-Mei Xiao (Medicine, HKU)
– Youling Guo (Psychiatry, HKU)
• Co-Investigators
– Patrick Kwan (Medicine, CUHK;
Medicine, Melbourne)
– Pak Sham (Psychiatry, HKU)
– Stacey Cherny (Psychiatry, HKU)
– Batool Haerian (Pharm., U. Malaya)
 Funded by HK grants
 HKU 762308M
 HKU 774707M
 CUHK 446606M
 CUHK 460313
 CUHK Direct Grant 2011.1.096

3. Epilepsy
 A chronic brain disorder with recurrent seizures
 Neurological diseases are growing
• Increased faster in the last generation as a cause of disability
adjusted life years lost (DALYs) than any other major disease
category except AIDS.
• Epilepsy DALYs rose ~30% to ~17 million, largely due to
population aging.
• ~50 million people have epilepsy.
 Epilepsy drugs treat most patients, but ~1/3 still
have seizures.
 Genetics may reveal causes, leading to new
treatment or prevention.

4. An epileptic seizure
https://www.youtube.com/watch?v=Nds2U4CzvC4

5. Epilepsy
 Prevalence is about 1%. Seizures affect 3% at
some time during life.
 Causes include genetics or any kind of brain
damage (stroke, injury, infection, etc.).
 Increases disability and doubles risk of death in
20 years.

6. Causes of epilepsy
 Change in neurons induces positive feedback in
neuronal circuits.
 Injury or illness might trigger regeneration, but
with abnormalities.
 Genetic factors, such as mutations in neuronal
ion channels, may predispose neurons to low
thresholds of excitation.

7. Types of epilepsy
 Idiopathic: not due to another disease or injury.
A variety of different seizure types. May include
genetic causes. Usually appears in young.
About 20-30% of all epilepsy.
 Symptomatic: an identified cause, such as a
stroke or other brain injury. Some may also
have genetic causes.
 Cryptogenic: seems like symptomatic, but
cause cannot be seen
 Unclassifiable

8. Genetics of epilepsy
 Idiopathic most commonly has a genetic cause,
thus is most studied. But other types are more
common and are less effectively treated.
 Why do some people with damage get epilepsy,
but not others? Is there a genetic propensity in
some people?

9. Role of genetics of epilepsy
 Generalized epilepsy syndromes: 80%
concordance among identical twins.
 Febrile seizures: 58% concordance.
 Partial epilepsies: 36%.
 Thus, epilepsy has a genetic component.

10. Genetic causes of epilepsy
 Mutations in some neuronal ion channels cause
some forms of idiopathic epilepsy.
• Sodium channels
• Nicotinic acetylcholine receptors
• Potassium channels
• Calcium chanels
• Chloride channels
• GABA receptors

11. Genetic causes of epilepsy
 Mutations in several genes of unknown function
can also cause idiopathic epilepsy.
 The genetic causes of most cases of idiopathic
epilepsy are still unknown.
 Mutations in many genes cause syndromes that
include epilepsy as one element.
• Mitochondrial genes
• Metabolic genes
• Brain development genes

12. Genetics of epilepsy types
Classification:
 Idiopathic epilepsy
• Strong genetic basis based on
family history
• Partly hereditary: the risk of
epilepsy among first-degree
relatives of idiopathic generalized
epilepsy (IGE) patients is 8-12%
Symptomatic epilepsy
• More common, develops after a wide
variety of brain insults/lesions
Most epilepsy genetics studies were of idiopathic
Finding genes for epilepsy
• Family studies
• Candidate gene studies
• Genome-wide association studies (GWAS)

13. Spectrum of genetic variants

14.  Rare, small variants (mutations)
• High penetrance
• Often identified in families
• Often in children
 Large variants (copy number variants)
 Common, small variants (polymorphisms)
• Low penetrance, but affect risk
• Common, thus might contribute to disease burden of
population as much as rare mutations
• Methods
 Candidate genes
 Genome-wide association studies (GWAS)
Spectrum of genetic variants

15. Mutations
Many are in ion channel genes
• SCN1A: voltage-gated sodium channel
• KCNQ2: potassium channel
• GABAA: GABAA receptor
• CHRNA4: acetylcholine receptor

16. SCN1A
 The most commonly mutated epilepsy gene
 Many different missense mutations can cause
epilepsy
 But many missense mutations do not cause
epilepsy
 Truncations almost anywhere can cause
epilepsy
 Different mutations can cause different
phenotypes

17. SCN1A mutations in epilepsy
Catterall W A et al. J. Neurosci. 2008;28:11768-11777
©2008 by Society for Neuroscience
Missense:
Truncation:

18. Candidate gene study: SCN
 Since SCN1A mutations can cause epilepsy, do
polymorphisms in SCN1A or other voltage-
gated sodium channel genes modify epilepsy
risk?
 We performed a case-control study
 828 epilepsy patients and 848 controls
 Selected 28 SNPs tagging SCN1A, SCN2A,
SCN3A, SCN1B, and SCN2B
 Genotyped by Sequenom MassArray

19. 0%
20%
40%
60%
80%
100%
GG
GA
AA
0%
20%
40%
60%
80%
100%
G
A
OR=0.84, p=0.02, for G vs. A OR=0.75, p=0.02, for GG vs. AA
SCN1A IVS5N+5G>A (rs3812718) was
associated with epilepsy
Candidate gene study: SCN

20. Adding Malaysians doubled the number
of subjects: 1529 patients and 1935
controls
IVS5N+5G>A was associated with
epilepsy
• G vs. A: OR=0.85, p=0.0009
• GG vs. AA: OR=0.73, p=0.003
Candidate gene study: SCN

21. Meta-analysis of epilepsy and
IVS5N+5G>A to confirm: p=0.002
Candidate gene study: SCN

22. Meta-analysis of epilepsy and
IVS5N+5G>A to confirm: p=0.002
Candidate gene study: SCN

23. SCN1A alternative splicing

24. SCN1A rs3812718: IVS5N+5G>A
 Allele G favors inclusion of 5N exon over 5A
exon.
• “N” for neonatal
• “A” for adult, which differs by a few amino acids
from N
 5N recovers faster from inactivation.
 Since SCN1A is preferentially expressed in
inhibitory (mostly GABAergic) neurons, G may
decrease epilepsy risk.
 Speaking of GABA…

25. GABAA receptor
 Voltage-gated GABAA receptor is the primary
mediator of synaptic inhibition in the brain
http://www.medscape.org/viewarticle/548383

26. GABAA receptor
 Mutations that reduce GABAA function can
cause epilepsy
 Missense or nonsense mutations
 Several genes
• GABRA1: childhood absence or juvenile
myoclonic
• GABRB3: childhood absence
• GABRG2: childhood absence, GEFS+, or Dravet

27.  488 symptomatic epilepsy patients + 2844
controls
 Genotyped rs211037 (c588C>T), in exon 5
Candidate gene study: GABRG2

28.  T allele
• Odds ratio=0.82
• P=0.005
 TT genotype (vs. CC)
• Odds ratio=0.65
• P=0.002
 Mechanism
• Not known
• Maybe in linkage disequilibrium with variant that
affects expression
Candidate gene study: GABRG2

29.  Genome-wide association studies (GWAS) of
idiopathic epilepsy
• SNPs in or near biologically plausible genes
 SCN1A
 CHRM3: muscarinic acetylcholine receptor
 VRK2: in a chromosomal deletion causing epilepsy
 ZEB2: mutations cause a syndrome including epilepsy
• Need confirmation in other studies and ethnic
groups
• More GWAS are being done and will be meta-
analyzed to find more variants and genes
GWAS of epilepsy

30. Cause of epilepsy
Those GWAS examined idiopathic epilepsy
What about symptomatic epilepsy?
• But why look for genetics if due to lesions?
 Not everyone with brain lesion gets epilepsy:
susceptibility factors.
 Analogy to other common diseases: complex

31. GWAS of epilepsy
We conducted a genome-wide
association study of non-idiopathic
epilepsy
2-stage design
• Stage 1: many SNPs in some of the patients
and controls
• Stage 2: top SNPs in some of the patients and
controls

32. Stage 1
 Subjects
• Han Chinese
• Patients: 504 symptomatic epilepsy patients from HK
• Controls: 2947 controls (1947 HK + 1000 Taiwan)
 SNPs
• Illumina HumanHap 610-Quad or 550-Duo BeadChips
• Genotyped at deCODE Genetics in Iceland

33. Stage 1 quality control
 Subjects
• Genotyping call rate > 95%
• Heterozygosity not too high
• Not related to or duplicated by other subjects
• Genotyped sex consistent with phenotype data
• Not outlier in multidimensional scaling
 Genotyping SNPs
• Genotyping call rate > 95%
• Minor allele frequency > 1%
• Hardy-Weinberg equilibrium P ≥ 0.0001
 Used 488 patients, 2875 controls, and 461,024
SNPs

34. Epilepsy patients in stage 1
Age 40±19 yrs
Sex 54% male
Phenotype
Stroke 97
Mesial temporal sclerosis 88
Infection 77
Vascular malformation 44
Cortical malformation 41
Others 38
Tumor 36
Trauma 27
Perinatal insults 20
Focal atrophy 16
Mental retardation 16
Total: Symptomatic 488

35. Stage 2
 Subjects
• Han Chinese
• Patients: 583 symptomatic and cryptogenic
epilepsy patients from HK
• Controls: 497 controls (HK Red Cross blood
donors)
 SNPs
• Stage 1 SNPs with lowest P values
• Only 1 SNP chosen for each 100 kb stretch
• Genotyped 86 and analyzed 82 after QC
• Used Sequenom MassARRAY at HKU Genome
Research Centre

36. GWAS results
b
Manhattan plot

37. Stage 1 Stage 2 Both
OR P-value OR P-value P-value
Gene Function
CAMSAP1L1 0.61 6.8×10-6 0.67 0.00038 1.0×10-8
Cytoskeletal protein; inhibits neurite extension;
expressed mainly in brain.
SNAR-H 1.48 2.4×10-5 1.16 0.026 5.3×10-5
RNA
KDM3A 1.68 5.4×10-5 1.50 0.033 4.9×10-5 Histone modifier
ERBB4 0.73 0.00016 0.86 0.048 5.8×10-5 Inhibitory synapse formation; mutations can cause
epilepsy.
SPEF2 1.47 0.00013 1.33 0.022 1.8×10-5 Motif like CAMSAP1L1; abundant in frontal cortex.
KCND2 0.76 0.00012 0.89 0.095 0.00013 Neuronal ion channel; mutations can cause epilepsy.
DSCAM 1.78 1.8×10-5 1.61 0.021 1.7×10-5
Regulates neuron connectivity and coordinated firing.
GWAS results

38. Genes identified in GWAS
SNAR-H KDM3A

39. Genes identified in GWAS
ERBB4 SPEF2

40. Genes identified in GWAS
KCND2 DSCAM

41. a b
CAMSAP1L1
rs2292096 G allele:
18% in controls,
12% in symptomatic epilepsy

42.  A cytoskeletal protein
 Calmodulin regulated spectrin-associated protein 1-like 1
What is CAMSAP1L1?
Baines et al. Mol Biol Evol 26:2005
 CH: calponin homology
 CKK: binds microtubules

43.  CKK fragment overexpression inhibits neurite
growth. Why?
 CAMSAP orthologue caps microtubule minus ends &
blocks microtubule movement
 By contrast, SCG10 destabilizes ─ends & increases
neurite growth. Counterintuitive?
What does CAMSAP1L1 do?
Hoogenraad et al. Trends in Cell Biology 19:669

44.  Stabilize neurites in mature neurons?
 Prevent excess growth during repair of damage?
• But growth & repair are good, right?
• Too much growth might form ectopic connections: short
circuits & seizures
+
+
What does CAMSAP1L1 do?

45. +
+
 Stabilize neurites in mature neurons?
 Prevent excess growth during repair of damage?
• But growth & repair are good, right?
• Too much growth might form ectopic connections: short
circuits & seizures
What does CAMSAP1L1 do?

46. +
 Stabilize neurites in mature neurons?
 Prevent excess growth during repair of damage?
• But growth & repair are good, right?
• Too much growth might form ectopic connections: short
circuits & seizures
What does CAMSAP1L1 do?

47. +
+
 Stabilize neurites in mature neurons?
 Prevent excess growth during repair of damage?
• But growth & repair are good, right?
• Too much growth might form ectopic connections: short
circuits & seizures
What does CAMSAP1L1 do?

48. +
+
+
+
+
 Stabilize neurites in mature neurons?
 Prevent excess growth during repair of damage?
• But growth & repair are good, right?
• Too much growth might form ectopic connections: short
circuits & seizures
What does CAMSAP1L1 do?

49. +
+
+
+
+
 Stabilize neurites in mature neurons?
 Prevent excess growth during repair of damage?
• But growth & repair are good, right?
• Too much growth might form ectopic connections: short
circuits & seizures
What does CAMSAP1L1 do?

50.  Hypothesis: CAMSAP1L1 variant → ↓expression
→ ↑neurites → ↑epilepsy risk
• rs2292096 A allele: higher epilepsy risk
• Do brains with A allele have ↓CAMSAP1L1?
• Do cells with ↓CAMSAP1L1 have ↑neurites?
 Methods
• Brain tissue from epilepsy surgery
 mRNA
 DNA
• SH-SY5Y neuroblastoma cells
 siRNA → ↓CAMSAP1L1
 Microscopy to count neurites
CAMSAP1L1 hypothesis

51. Expression vs. genotype
Temporal lobe
A+ genotypes: p=0.024
Hippocampus
A+ genotypes: p=0.173

52. Localization

53. siRNA
2 3 4 5

54. Neurites
*
*
*
*
*

55. ERBB4
 Our GWAS: rs13021324 C allele decreases epilepsy risk
 Tyrosine kinase receptor
 Decreased ERBB4 expression in temporal lobe epilepsy
(60%)
 Downregulation of signaling between NRG1 (neuregulin)
and ERBB4 in interneurons in epilepsy
 Erbb4−/− mice more susceptible to induced epilepsy
 One case with early myoclonic epilepsy has a disruption
of gene ERBB4
 Is rs13021324 C allele associated with increased ERBB4
expression?
Li et al. Nat Neurosci (2012), 15(2), 267-73.

56. Hippocampus
Mann-Whitney
test: P=0.28
ERBB4 RNA vs. genotype

57. Temporal lobe
Mann-Whitney
test: P=0.22
ERBB4 RNA vs. genotype

58. ERBB4
 rs13021324 C allele shows trend toward
association with increased ERBB4 expression
 Need more samples to confirm
 Mechanism not known: maybe in LD with
variant that affects expression

59.  Hypothesis: CAMSAP1L1 variant → ↓expression →
↑neurites → ↑epilepsy risk
• rs2292096 A allele: higher epilepsy risk
• Brains with A allele have ↓CAMSAP1L1
• Cells with ↓CAMSAP1L1 have ↑neurites
 SCN1A IVS5N+5G>A associated with epilepsy
 GABRG2 rs211037 associated with epilepsy
 Further research
• Studying roles of the proteins in neurons and epilepsy
• Finding other proteins in pathways with these proteins
• Treating animal models of post-injury epilepsy
 Implications
• Screening for brain damage patients at risk
• Developing treatments blocking these pathways
Conclusions

60. The End
Thank you