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CFERV 2019 Wenshu

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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.

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CFERV 2019 Wenshu

  1. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 13 Functional Evaluation of 6 novel GRIN2D variants © Wenshu XiangWei & Yuwu Jiang
  14. 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. 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. 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. 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. 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. 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. 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. 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. 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

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