Genetic variation in GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits has been associated with a spectrum of neurological and neuropsychiatric disorders. This talk will focus on clinical phenotype, functional consequences, and potential rescue pharmacology of disease-associated GRIN2D missense variants. The GRIN2D variants are located in the pre-M1 helix, agonist-binding domain, transmembrane domain M3, and the intracellular CTD. Developmental and epileptic encephalopathy (DEE) is the unifying phenotype across all 13 patients. The seizure types ranged from focal seizures, atypical absence seizures, tonic or atonic seizures, to epileptic spasms. Eight patients showed certain degree of hypotonia and movement disorders, three patients have autistic behavior and one patient showed symptoms of ADHD. Functional analysis in vitro on six novel GRIN2D variants reveals that that all six variants decreased receptor surface expression, which may underline certain shared clinical symptoms. Three variants (Leu670Phe, Ala675Thr, Ala678Asp) showed significantly enhanced agonist potency, and/or increased channel open probability, while the other three (Ser573Phe, Ser1271Phe, Arg1313Trp) presented reduced sensitivity to endogenous protons and decreased channel open probability. Three variants (Ser573Phe, Ala675Thr, Ala678Asp) significantly decrease current amplitude. GluN2D-Leu670Phe prolongs synaptic-like response time course and increases charge transfer. GluN2D-Ala678Asp transfection significantly reduced cell viability of cultured cortical neurons. In addition, a set of FDA-approved NMDAR channel blockers were evaluated for their ability to rescue functional changes of mutant receptors. This work suggests the importance of functional and biochemical validation for each individual variant. Rescue pharmacology in vitro may provide potential benefit of precision medicine for a subset of severe pediatric neurodevelopmental diseases.

1. Wenshu XiangWei and Yuwu Jiang
Department of Pediatric and Epilepsy Center,
Peking University First Hospital
9/13/2019
Clinical Features and Mechanism of GRIN2D-
related Developmental and Epileptic
Encephalopathy
© Wenshu XiangWei & Yuwu Jiang

2. Department of Pediatric, Peking University First Hospital
2
 Established in 1929
 One of the biggest epilepsy centers for children in China
 Outpatient: 30,000 to 40,000 every year
 Inpatient: 208 beds in total (40 beds of neurology and 20 beds of VEEG
recording)
 Over 1,500 genetic tests every year
 Enrolled in a network with 23 hospitals
across China
© Wenshu XiangWei & Yuwu Jiang

3. Positive % of Gene Panel
3
XiangWei et al., Unpublished data
Detectionnumber(N)
• Design for patients with epilepsy and intellectual/developmental delay
• Include 480 epilepsy-related gene
• Detection Rate : 22% (541/2462)
• 24% (129/541) in sodium channel genes, 10% (52/541) in potassium
channel genes, 3% (15/541) in GABA receptor genes and 3% (15/541) in
glutamate receptor genes
© Wenshu XiangWei & Yuwu Jiang

4. GRIN variants
4
XiangWei et al., Unpublished data
Gene c. p. Phenotype Gene c. p. Phenotype
GRIN1 c.1100G>A p.Arg367Gln EPI/ID GRIN2A c.2191G>A p.Asp731Asn EPI/ID
GRIN1 c.2444G>C p.Arg815Pro EPI/ID GRIN2B c.1619G>A p.Arg540His EPI/ID
GRIN2A c.1497+1G>A EPI/ID GRIN2B c.1907C>T p.Ala636Val EPI/ID
GRIN2A c.82G>T p.Glu28* EPI/ID GRIN2B c.2038C>A p.Pro680Thr EPI/ID
GRIN2A c.890G>T p.Gly297Val EPI/ID GRIN2B c.1177C>T p.R393X EPI/ID
GRIN2A c.2279G>T p.Gly760Val EPI/ID GRIN2B c.1985A>C p.Gln662Pro EPI/ID
GRIN2A c.3491C>T p.Thr1164Met EPI/ID GRIN2D c.1718C>T p.Ser573Phe DEE
GRIN2A c.869C>T p.A290V EOEE GRIN2D c.1999G>A p.Val667Ile DEE
GRIN2A p.C455Y ABPE GRIN2D c.2033C>A p.Ala678Asp DEE
GRIN2A c.2107C>T p.Q703X EAS GRIN3A c.3081C>A p.Tyr1027* DEE
GRIN2A p.G760S LKS GRIN3B c.3113G>T p.Arg1038Leu DEE
GRIN2A p.C231R ABPE GRIN3B c.2205delC p.Lys738Serfs
*25
DEE
GRIN2A p.D1385Y ABPE
© Wenshu XiangWei & Yuwu Jiang

5. 16 patients with 12 GRIN2D Variants
5Tsuchida N et al., Clin Genet. (2018) Li et al. Am J Hum Genet (2016)
XiangWei et al., Brain (2019 XiangWei et al., Unpublished data
Variants Location Case Phenotype
c.1345G>A D449N ABD (S1) 1 epi, delay
c.1718C>T S573F pre-M1 1 epi, delay
c.1999G>A V667I M3 4 epi, delay
c.2008C>T L670F M3 2 epi, delay
c.2023G>A A675T M3 1 epi, delay
c.2033C>A A678D M3 1 epi, delay
c.2043G>C M681I M3 1 epi, delay
c.2044A>C I682L M3 1 epi, delay
c.2080A>C S694R ABD (S2) 1 epi, delay
c.2533A>G M824V M4 1 delay
c.3812C>T S1271L CTD 1 epi, delay
c.3937C>T R1313W CTD 1 epi, delay
Glutamate
LBD
Membrane
Out
In
M1
ABD
ATD
M4
M2
CTD
© Wenshu XiangWei & Yuwu Jiang

6. Phenotype of GRIN2D Variants
6
 Gender
7 male and 9 female
 Age
9mo to 34yo, 1 patient (A675T) passed away before 2yo
 Family history and perinatal period
unremarkable
 Diagnosis
• 94% (15/16) of developmental and epileptic encephalopathy (DEE)
• Only 1 patients (M824V) only has severe developmental delay, no seizure until last
follow-up (1y, 4mo)
© Wenshu XiangWei & Yuwu Jiang

7. Phenotype: Seizure onset age
7
15/16 patients have epilepsy
• Onset age from 1mo to 3y, 5mo
• 75% (12/16) onset before 1yo
Before 1yo
[PERCENTAG
E]
No seizure
[PERCENTAG
E]
After 1yo
[PERCENTAG
E]
© Wenshu XiangWei & Yuwu Jiang

8. 8
 focal
 myoclonic
 GTCS
 epileptic spasms
 afebrile
 atypical absence
 5 patients with complexed seizure type
Focal
[PERCENTA
GE]
myoclonic
[PERCENTA
GE]
GTCS
[PERCENTA
GE]
Spasms
19%
atypical
absence
9%
afebrile
10%
Phenotype: Seizure type
© Wenshu XiangWei & Yuwu Jiang

9. 9
No
response
[PERCENTA
GE]
Seizure free
[PERCENTA
GE]
With no
formal
therapy
[PERCENTA
GE]
Phenotype: Response to anti-epileptic Drugs
 2/15 with no formal therapy
 11/15 were no response
 2/15 were seizure free:
• A678D: valproate, levetiracetam and clonazepam
• V667I: memantine, IVIG, oral steroids and Mg
© Wenshu XiangWei & Yuwu Jiang

10. Phenotype: EEG
10
All 16 patients had abnormal EEG
p.(S1271L) Showed hypsarrhythmia
p.(A678D) Showed spike and spike-wave
in bilateral Rolandic region and mid-line
multifo
cal
spike
62%
[CATEG
ORY
NAME]
[PERCE
NTAGE]
[CATEG
ORY
NAME]
[PERCE
NTAGE]
© Wenshu XiangWei & Yuwu Jiang

11. Phenotype: MRI
11
 9/15 patients were normal
 6/15 patients showed mild cerebral atrophy
p.(L670F) showed cerebral atrophy (2-year-old)(5-month-old)
© Wenshu XiangWei & Yuwu Jiang

12. Phenotype: developmental delay
12
 All patients with intellectual/developmental delay (ID/DD)
• 1/16 mild
• 15/16 severe (DQ<40)
 Hypotonia and movement disorder
• 10/16 had mild to severe hypotonia
• 3/16 had hypertonia
 Other neurological features
• Poor sleep, autistic behavior, ADHD, visual impairment, microcephaly
© Wenshu XiangWei & Yuwu Jiang

13. 13
Functional Evaluation of 6 novel GRIN2D variants
© Wenshu XiangWei & Yuwu Jiang

14. 1E-3 0.01 0.1 1 10 100
0
20
40
60
80
100
MaximalResponse,%
Glutamate, mM
WT 2D
S573F
A675T
A678D
L670F
S1271L
R1313W
Glutamate, EC50, µM
WT 2D 0.39 ± 0.02
S573F 0.31 ± 0.04*
L670F 0.12 ± 0.02* 3-fold ↑
A675T 0.02 ± 0.004* 19-fold ↑
A678D 0.03 ± 0.01* 13-fold ↑
S1271L 0.25 ± 0.03 NS
R1313W 0.31 ± 0.03 NS
N=11-30 oocytes
14
Effects of GluN2D variants on agonist potency
© Wenshu XiangWei & Yuwu Jiang

15. 0.01 0.1 1 10
0
20
40
60
80
100
Glycine, mM
MaximalResponse,%
WT 2D
S573F
A675T
A678D
L670F
S1271L
R1313W
0.01 0.1 1 10
0
20
40
60
80
100
D-Serine, mM
MaximalResponse,%
Glycine, EC50, µM D-serine, EC50, µM
WT 2D 0.12 ± 0.01 0.14 ± 0.01
S573F 0.09 ± 0.01 0.08 ± 0.005*
L670F 0.09 ± 0.02* 0.05 ± 0.01* 3-fold↑
A675T 0.04 ± 0.005* 3-fold ↑ 0.01 ± 0.002* 14-fold↑
A678D 0.06 ± 0.01* 2-fold↑ 0.04 ± 0.01* 3-fold↑
S1271L 0.24 ± 0.02* 2-fold↓ 0.13 ± 0.01
R1313W 0.23 ± 0.01* 2-fold↓ 0.15 ± 0.02
N=11-38 oocytes 15
WT 2D
S573F
A675T
A678D
L670F
S1271L
R1313W
Effects of GluN2D variants on agonist potency
© Wenshu XiangWei & Yuwu Jiang

16. 1 10 100 1000
0
20
40
60
80
100
Mg2+ , mM
MaximalResponse,%
WT 2D
S573F
A675T
A678D
L670F
S1271L
R1313W
IpH6.8/IpH7.6,%
*
**
0
20
40
60
80
**
Mg2+, IC50, µM Proton, %
WT 2D 158 ± 11 26 ± 0.7
S573F 91 ± 12* 33 ± 1.7*
L670F 199 ± 0.6 65 ± 3.0*
A675T 152 ± 11 49 ± 3.4*
A678D 160 ± 23 26 ± 2.4
S1271L 217 ± 0.8 25 ± 1.5
R1313W 190 ± 0.8 29 ± 2.3
N=8-38 oocytes 16
Effects of GluN2D variants on antagonist potency
© Wenshu XiangWei & Yuwu Jiang

17. CalculatedPOPEN
**
ND
0
0.01
0.02
**
0.2
0.5
0.8
Calculated POPEN
WT 2D 0.0067 ± 0.0003
S573F 0.0016 ± 0.0002* 4-fold
L670F 0.36 ± 0.01* 54-fold
A675T ND
A678D 0.20 ± 0.02* 30-fold
S1271L 0.0032 ± 0.0003 2-fold
R1313W 0.0025 ± 0.0003 2-fold
N=40-112 oocytes
17
Effects of GluN2D variants on channel open probability
© Wenshu XiangWei & Yuwu Jiang

18. 18
Effects of GluN2D variants on current amplitude and response time course
15 sec
0.15 normalized
WT 2D L670Fglutamate
0
20
40
60
CurrentAmplitude,pA/pF
*
** **
Current amplitude, pA/pF Deactivation tW, ms Charge transfer, ms × pA/pF N
WT 2D 29 ± 2.9 5,500 ± 260 150,000 16
S573F 12 ± 3.6* 4,000 ± 360 53,000 10
L670F 21 ± 5.5 37,000 ± 9,000* 790,000* 10
A675T 0.07 ± 0.03* ND ND 15
A678D 0.12 ± 0.04* ND ND 14
S1271L 17 ± 2.8 5,000 ± 430 86,000 9
R1313W 20 ± 5.6 4,900 ± 230 96,000 12
© Wenshu XiangWei & Yuwu Jiang

19. 19
Surface/Total, % Total, %
WT 2D 1 1
S573F 0.60 ± 0.13* 0.57 ± 0.1*
L670F 0.62 ± 0.09* 0.57 ± 0.09**
A675T 0.21 ± 0.02** 0.68 ± 0.09**
A678D 0.35 ± 0.06* 0.67 ± 0.07**
S1271L 0.69 ± 0.16* 0.58 ± 0.08**
R1313W 0.33 ± 0.13** 0.18 ± 0.08**
N=4-14 times
Effects of GluN2D variants on NMDAR traffickingSurfaceexpress(%ofWT)
0
40
80
120
WT 2D
*
*
*
**
**
**
0
40
80
120
© Wenshu XiangWei & Yuwu Jiang

20. The effects of FDA-approved NMDAR channel blockers
20
0.1 1 10 100
0
20
40
60
80
100
MaximalResponse,%
Memantine, mM
WT 2D
A675T
A678D
L670F
1 10 100
0
20
40
60
80
100
Dextromethorphan, mM
WT 2D
A675T
A678D
L670F
0.1 1 10 100
0
20
40
60
80
100
Ketamine, mM
WT 2D
A675T
A678D
L670F
IC50, µM Memantine Dextromethorphan Ketamine
WT 2D 2.5 ± 0.5 5.1 ± 0.7 4.5 ± 0.6
L670F >150* >300* >2,000*
A675T 11 ± 0.9* 10 ± 0.6* 25 ± 3.0*
A678D 5.8 ± 0.9* 6.8 ± 0.7 5.1 ± 0.8
N=11-19 oocytes, inhibition % =11-94
© Wenshu XiangWei & Yuwu Jiang

21. Summary
21
• GRIN2D variants are involved in a subset of patients with
developmental epileptic encephalopathy
• Variants in pre-M1 (S573F) and CTD (S1271L and R1313W) show
modest change in agonist and antagonist potency, but reduce
channel open probability and surface expression
• Variants in M3 (L670F, A675T, A678D) increase channel open
probability, reduce agonist potency, decrease receptor cell surface
expression
• Dextromethorphan and ketamine may be effective in patients with
A678D variant
© Wenshu XiangWei & Yuwu Jiang

22. Acknowledgement
22
Supporting from all my patients and their families
Department of Pediatrics,
Peking University First Hospital
Yuwu Jiang
Ye Wu
Yuehua Zhang
Xinhua Bao
Zhixian Yang
Taoyun Ji
CFERV, Emory University School of Medicine
Stephen F. Traynelis
Hongjie Yuan
Scott Myers
Subh Bhattacharya
Yuchen Xu
Jing Zhang
Suhkan Kim
Kai Gao
Jie Zhang
Nana Liu
Huifang Yan
Ying Yang
All Collaborators (on GRIN2D project)
Johannes R. Lemke (Leipzig, Germany)
Annapurna Poduri (Harward, US)
Elias Aizenman (Pittsburgh, US)
Gaetan Lesca (Lyon, France)
Jean-Marie Cuisset (Lille, France)
Katherine L. Helbig (Philadelphia, US)
Dragan Marjanovic (Dianalund, Denmark)
Steffen Syrbe (Heidelberg, Germany)
An-Sofie Schoonjans, Nina Dirkx (Antwerp, Belgium)
Funding CSC, NSF
Disclosures None
© Wenshu XiangWei & Yuwu Jiang