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Supplementary Materials for
L-Serine dietary supplementation is associated with clinical improvement of
loss-of-function GRIN2B-related pediatric encephalopathy
David Soto, Mireia Olivella, Cristina Grau, Judith Armstrong, Clara Alcon, Xavier Gasull, Ana Santos-Gómez, Sílvia Locubiche, Macarena Gómez de Salazar, Roberto García-Díaz, Esther Gratacòs-Batlle, David Ramos-Vicente,
Emeline Chu-Van, Benoit Colsch, Víctor Fernández-Dueñas, Francisco Ciruela, Àlex Bayés, Carlos Sindreu, Anna López-Sala, Àngels García-Cazorla*, Xavier Altafaj*
*Corresponding author. Email: [email protected] (X.A.); [email protected] (À.G.-C.).
Published 18 June 2019, Sci. Signal. 12, eaaw0936 (2019)
DOI: 10.1126/scisignal.aaw0936
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Fig. S1. Protein interactions and cellular trafficking of GluN2Bwt- and GluN2B(P553T)-containing NMDARs. Fig. S2. Altered biophysical properties of heterotrimeric GluN1-GluN2A-GluN2B(P553T) NMDARs. Fig. S3. GluN2B(P553T) mutation alters GluA1 abundance in hippocampal neurons. Fig. S4. Alignment of eumetazoan iGluRs showing the residues conservation of Pro553 and Phe653. Table S1. Untargeted analysis of plasma sphingolipid profile in the GRIN2B(P553T) patient before and after L-serine dietary supplementation. References (78, 79)
Fig. S1. Protein interactions and cellular trafficking of GluN2Bwt- and GluN2B(P553T)-containing NMDARs. (A) Western blot analysis of heterologous
expression of wildtype (first lane) and (P553T) mutant GFP-GluN2B (second lane) in equal amounts of proteins (actin loads) extracted from transiently transfected HEK-293T cells. (B) Representative western blot analysis of co-immunoprecipitation experiments from HEK-293T cells transiently co-transfected with HA-GluN2A, GluN1 and either GFP-GluN2Bwt or GFP-GluN2B(P553T). The co-immunoprecipitation was performed either with anti-GFP (pulling-down GFP-GluN2B constructs products and their interacting proteins) or IgGs (negative control). Interaction studies showed the ability of GFP-tagged GluN2B(P553T) subunit to interact with HA-GluN2A and GluN1 subunits, similarly to GluN2Bwt. Bar graph representing the densitometric analysis of immunoprecipitated proteins (relative to immunoprecipitated GFP-GluN2B for GluN2A and GluN1 subunits; relative to GFP-GluN2B load for GluN2B subunit) (mean ± SEM densitometric of three independent experiments; white bars: GluN2Bwt condition; red bars: GluN2B(P553T) condition). (C) Immunofluorescent analysis (left) of cell surface expression (red channel) and total expression (green channel) of wildtype (left panels) and mutant GFP-GluN2B (right panels) in COS-7 cells. ar graph (right) shows the relative surface to total expression of GFP-GluN2B constructs in COS-7 cells (n=35-38 cells per condition, from 3 independent experiments; *p=0.007, Student's t test). (D) Time-course analysis of GFP-GluN2B cell surface expression (green channel) and intracellular expression (red channel) of wildtype (left panels) and mutant GFP-GluN2B (right panels), in primary cortical neuronal cultures transiently transfected. Primary cultures were transfected at day in vitro 4 (DIV4) or DIV7, and immunofluorescence analysis of surface (green channel) and intracellular (red channel) expression was performed at DIV6, DIV11 and DIV16. Bar graph (bottom) shows the ability of mutant GluN2B(P553T)-containing NMDARs to reach the plasma membrane of the dendritic processes (insets) of transfected cortical neurons (n=25-62 cells per condition, from 3-4 independent experiments; *p=0.026, ns, non-significant; Student's t test). Scale bar = 40 µm (low magnification) or 3 µm (dendrites).
Fig. S2. Altered biophysical properties of heterotrimeric GluN1-GluN2A-GluN2B(P553T) NMDARs. (A) Representative traces evoked by 1mM glutamate plus 1μM glycine (0.5 s; −60mV) in HEK-293T cells expressing triheteromeric GluN1-GluN2A-GluN2Bwt (2A-2B, left traces) or GluN1-GluN2A-2B(P553T) (2A-2B(P553T), right traces) in the absence (black traces) or in the presence (red traces) of 100 μM D-serine. (B) Normalized peak currents (−pA/pF) for GluN2A-2B- and GluN2A-2B(P553T)-expressing cells with single experiment values superimposed (open circles; p>0.05; Mann Whitney U-test; n=18 and 13, respectively). (C) Percentage of current increment in the presence of 100 μM D-serine, for GluN2A-2B and GluN2A-2B(P553T) expressing cells with single experiment values superimposed (open circles; p<0.05; Mann Whitney U-test; n=14 and 13, respectively). (D) Representative whole-cell currents evoked by 1mM glutamate plus 1μM glycine from HEK-293T cells expressing GluN2A-2B (black trace) or GluN2A-2B(P553T) (red trace), showing the faster kinetics of triheteromeric NMDARs containing GluN2B(P553T) subunit. Responses have been peak-scaled for comparison purposes. (E) Magnification of the tail deactivating currents (shown in panel D) after glutamate and glycine removal. Traces (aligned at the coagonists removal time-point) showed a faster deactivating kinetics of mutant triheteromeric NMDARs (0.14s for depicted cell, red trace) compared with GluN2Bwt-containing triheteromers (0.29s for depicted cell, black trace). Fits are shown overlapped in green. (F) Magnification of triheteromeric NMDARs desensitization (shown in panel D). Both currents have been aligned at the peak current. GluN2B(P553T)-containing receptors show a faster desensitization time constant (0.12s for depicted cell, red trace) compared with GluN2Bwt-containing triheteromers (0.38 s for depicted cell, black trace). Fits are shown in green. (G) Bar graph representing the deactivation time constant fitted from tail currents for triheteromeric GluN2A-2B and GluN2A-2B(P553T). GluN2B(P553T) mutation accelerates channel closure after coagonists
removal (0.30 ± 0.02s for GluN2A-2B vs. 0.21 ± 0.02s for GluN2A-2B(P553T); p<0.01; Mann-Whitney U-test; n=17 and 14, respectively). Single experiment values are shown as open circles for each condition. (H) Bar graph representing the average desensitization time constant for GluN2A-2B- and GluN2A-2B(P553T)-expressing cells, measured in the presence of the agonists (as shown in panel F). P553T mutation accelerates desensitization of triheteromeric NMDARs (0.30 ± 0.02 s for GluN2A-2B vs. 0.15 ± 0.02 s for GluN2A-2B(P553T); p<0.0001; Mann-Whitney U-test; n=17 and 14 respectively). Single experiments values are shown as open circles for each condition.
Fig. S3. GluN2B(P553T) mutation alters GluA1 abundance in hippocampal neurons. Time course of surface expression of the AMPAR GluA1 subunit in primary hippocampal neurons transfected GFP-GluN2Bwt or GFP-GluN2B(P553T). Immunofluorescent analysis of surface GluA1 expression (red channel) was performed at different developmental stages (DIV6, 11 and 16) in GFP-GluN2B transfected neurons (green channel). Bar graph representing surface expression levels of GluA1. Note the significant increase of GluA1 surface levels of neurons expressing mutant GFP-GluN2B(P553T) at DIV11 (n=16-43 dendrites per condition, from 3 independent experiments; ***p < 0.001; ns, non-significant; Student's t test).
Fig. S4. Alignment of eumetazoan iGluRs showing the residues conservation of Pro553 and Phe653. The alignment includes 147 protein sequences from members of diverse phylogenetic groups of the iGluR protein family. Due to space limitation, the alignment is divided into two columns. The alignment includes sequences from bilaterian and cnidarian species (eumetazoans). Higher amino acid conservation is represented by increasing
GluN2A_Hsa
GluN2B_Hsa
GluN2C_Hsa
GluN2D_Hsa
GluN2ADalpha_Bbe
GluN2ADbeta_Bbe
GluN2ADgamma_Bbe
GluN2ADdelta_Bbe
GluN2ADalpha_Bla
GluN2ADbeta_Bla
GluN2ADalpha_Sko
GluN2ADalpha_Lgi
GluN2ADbeta_Lgi
GluN2ADgamma_Lgi
GluN2AD_Cte
GluN2ADalpha_Sma
GluN2ADbeta_Sma
GluN2ADgamma_Sma
GluN2AD_Ame
GluN1_Hsa
GluN1_Bbe
GluN1_Bla
GluN1alpha_Sko
GluN1beta_Sko
GluN1_Apl
GluN1_Lgi
GluN1_Cte
GluN1_Sma
GluN1_Ame
GluN1_Pca
GluN1_Nve
GluN3A_Hsa
GluN3B_Hsa
GluN3AB_Bbe
GluN3ABalpha_Sko
GluN3ABbeta_Sko
GluN3AB_Apl
GluN3AB_Lgi
GluN3AB_Cte
GluN3AB_Sma
GluN3AB_Ame
GluN23_Nve
GluNCni2_Nve
GluNCni3_Nve
GluNCni4_Nve
GluNCni5_Nve
GluA1_Hsa
GluA2_Hsa
GluA3_Hsa
GluA4_Hsa
GluA14alpha_Bbe
GluA14beta_Bbe
GluA14alpha_Bla
GluA14beta_Bla
GluA14_Sko
GluA14alpha_Spu
GluA14beta_Spu
GluA14alpha_Apl
GluA14beta_Apl
GluA14alpha_Lgi
GluA14beta_Lgi
GluA14gamma_Lgi
GluA14delta_Lgi
GluA14epsilon_Lgi
GluA14alpha_Cte
GluA14beta_Cte
GluA14gamma_Cte
GluA14delta_Cte
GluA14alpha_Sma
GluA14beta_Sma
GluA14gamma_Sma
GluA14_Ame
GluK1_Hsa
GluK2_Hsa
GluK3_Hsa
GluK4_Hsa
GluK5_Hsa
GluK15alpha_Bbe
GluK15beta_Bbe
GluK15alpha_Bla
GluK15betaA_Bla
GluK15betaB_Bla
GluK15_Sko
GluK15alpha_Spu
GluK15beta_Spu
GluK15alpha_Apl
GluK15beta_Apl
GluK15alpha_Lgi
GluK15beta_Lgi
GluK15_Cte
GluK15alpha_Sma
GluK15beta_Sma
GluK15gamma_Sma
GluK15delta_Sma
GluK15epsilon_Sma
GluK15alpha_Ame
GluK15beta_Ame
GluK15gamma_Ame
GluK15delta_Ame
GluK15epsilon_Ame
GluD1_Hsa
GluD2_Hsa
GluD12alpha_Bbe
GluD12beta_Bbe
GluD12gamma_Bbe
GluD12alpha_Bla
GluD12alpha_Sko
GluD12beta_Sko
GluD12_Spu
GluD12_Apl
GluD12alpha_Lgi
GluD12beta_Lgi
GluE1_Cin
GluE2_Cin
GluE1_Bbe
GluE2_Bbe
GluE3_Bbe
GluE4_Bbe
GluE5_Bbe
GluE6_Bbe
GluE7_Bbe
GluE8_Bbe
GluE1_Bla
GluE6_Bla
GluE1_Sko
GluE2_Sko
GluE1_Pfl
GluE2_Pfl
GluE1_Nve
GluE2_Nve
GluF1_Bbe
GluF2_Bbe
GluF1_Bla
GluF2_Bla
GluF1_Bfl
GluF2_Bfl
GluF3_Bfl
GluF4_Bfl
GluF_Sko
GluF_Pfl
GluF1_Spu
GluF2_Spu
GluF3_Spu
GluF_Apl
GluAkdf1_Nve
GluAkdf2_Nve
S A F L E P F S A S V A N L A A F M I - - - - Q E E
S A F L E P F S A D V A N L A A F M I - - - - Q E E
S A F L E P Y S P A V A N L A A F M I - - - - Q E Q
S A F L E P Y S P A V A N L A A F M I - - - - Q E E
T A F L E P F DWT F A N L A V F M I - - - - Q E Q
T A F L E P F D V F A A N L A A F M I - - - - Q E E
S A F L E P Y D A T L A N L A A F M I - - - - Q E Q
S A F L A P L D V WS A K L T A F M I - - - - L E E
T A F L E P F DWT F A N L A V F M I - - - - Q E Q
T A F L E P F D V F A A N L A A F M I - - - - Q E E
H A F L A P F D I A I G N L A A HM I - - - - H E E
T A F L E P Y D Y P A A N L A A F M I - - - - T K E
T A F L E P Y D Y P S A N L A A F M I - - - - T K E
K A F L E P Y D Y P S A N L A A F M I - - - - T K E
T A F L E P F D I L S A N L A A F M I - - - - T K E
K A F L E P F D T Y S A N L A A F M I - - - - T G E
K A F L E P F D T M S A N L A A F M I - - - - T R E
K A F L E P F D N I S A N L A A F M I - - - - T R E
T A F L E P F D T A S A N L A A F M I - - - - T R E
D S F MQ P F Q S T L A N L A A F L V - - - - L D R
D S F L Q P F Q S T L A N L A A F L V - - - - L D R
D S F L Q P F Q S T L A N L A A F L V - - - - L D R
T S F L Q P F E S A L A N L A A F L V - - - - L D R
A S F F Q P F E S A L A N L A A F L V - - - - L D R
L S F M R P F Q I S L A N L A A Y L V - - - - L D K
A S F L Q P F Q D T L A N L A A F L V - - - - L D R
A S F L Q P F Q D T L A N L A A F L V - - - - L D R
V S F L Q P F Q D T L A N L A A F L V - - - - L E R
V S F L Q P F S N T L A N L A A F L V - - - - L E R
T S F L Q P F Q D S L A N L A A F L V - - - - L D K
D S F L R P F Q I H L A N L A A F L V - - - - L D R
G A F MWP L HWT M A N L A A V M V - - - - G E K
G A F MWP L HWS T A N L A A V M V - - - - G D K
A S F M K P L DWSM A N L A A F M V - - - - D E K
G S F M E P L EWT M A N L A A F M V - - - - G E K
D S F L E P L DWS V A N L A A Y M A - - - - GQ T
G A F L A P L H P N V A N L A A F L A - - - - GQM
Q A F L E P F A T Q V A N L A A F I A - - - - G K H
Y A F M E P F D S WV A N L A A F L A - - - - G K N
H A F L L T F S P Y L A N I A R L F A - - - - G L F
F A F L F P F S P E L A N I A A L I A - - - - G L F
DG F L K P F K V S A A N L A A F M V - - - - L Q D
WN F Q D P F HWD L A N L T A F L L - - - - E D N
L A F I R P F D N S L A N L T A S L V - - - - Q E L
F A F F D P F T WQ L A N L T A H L V - - - - S D D
M D F M I P L S S Q L A E L A A F K V - - - - K E Q
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L D P L A Y E I A N L A A F L T - - - - V E R
F S F L H P L S H E I A N L A A F L T - - - - V E R
F S F M H P L S Y E I A N L A A F L T - - - - V E R
F S F F Q P F T N E I A N L A A F L T - - - - T Q S
F S F M H P L S Y E I A N L A A F L T - - - - V E R
F T F MQ P L A P E I A N L A A F L T - - - - A K R
F S F M D P L S Y E I A N L A A F L T - - - - V E R
F S F M E P L S S E I A N L A A H L T - - - - F M R
F S F M E P L S F Q I A N L A A F L T - - - - I E R
F S F M S P L A K Y V A N L A A F L T - - - - I E K
F S F MQ P L DM T V A N L A A F L T - - - - I E K
F S F M Y P L S Y E I A N L A A F L T - - - - V S R
F S F M Y P L S Y E I A N L A A F L T - - - - V S R
F S F M Y P L S T E I A N L A A F L T - - - - V A R
F S F M D P L S Y E I A N L A A F L T - - - - V E R
F S F M N P L S K E I A N L A A F L T - - - - V E R
L S F M K P L S I E I A N L A A V L T - - - - V E R
F S F M K P L SM E I A N L A A F L T - - - - V E R
F S F L N P L S K E I A N L A A F L T - - - - V E R
F S F L N P L S P D I
F S F L N P L S P D I
F S F L N P L S P D I
F S F L D P F S PG V
F S F L D P F S P A V
F A F L S P L S Y D I
F S F L N P L S Y D I
F A F L S P L S Y D I
F S F L N P L S Y D I
F S F L N P L S Y D I
F S F L S P L D F D I
F S F L N P L S F D I
F S F L N P L S P D V
F S F L N P L S F D V
F S F L N P L S F D I
F S F L N P L A I E I
F S F L S P L S V E V
F S F L N P L A I E I
F S F L N P F S E D V
F S F L S P F S V E V
F A F L N P F S E D V
WV F L A P F E SG V
F S F M N P L A I E I
F S F L S P L S L D V
F S F L S P L S T D V
F S F L S P F S AG V
F S F M N P L A V E I
F S F M N P L A I Q I
F S L F A P F D F A V
F A C L A P F D L S L
F A F L E P F N F Q V
F G F MG P F T P E V
L G F L G P F S V Y V
F A F L E P F N F Q V
F A F L Q P L R I S V
F A F M E P L S G P V
F A F L E P L D I K V
F A F L E P L H I K V
F R S F K P F T T N V
F K M F T P F A P V V
Y A F L E P F E T N L
YG F L R P F T WT L
F K F L D V F E P N L
F K F L E P F E I R L
F N F F G P L E K R L
M N F S R P F Q P E L
WG F V K P F E G R L
WG F I S P F QG E L
WG F I S P F QG E L
WG F V S P F Q A D L
F K F L D V F E P N L
WG F I S P F E G E L
F A F L D P F S Y D L
F Q F L E P F S T D L
F A F L D P F S Y D L
F R F A E P F S G D L
F R F L E P F K S D L
F A I L R P F R Y D L
F E F L A P F D N L V
L A F M T P L S P L M
F E F L A P F D I WV
L S F M T P L S PQM
L Q F L N P F S Y T V
L A F M T P L S P P M
F Q F L A P F S T T V
F Q F MG P F S V E V
F G I F T P F T F WV
F A F L T P YQ A P V
F V P V F P Y DWN V
F V P V F P F N I Y A
F R L T Y P F G I E V
F R I M Y P F G I E V
F A F L L P F D E K L
F A F L M P F Q K D L
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V Q R
A N L A A F L T - - - - V Q R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A T L A A F L T - - - - A G R
A N L A A F L M - - - - L E P
A T L A A F I T - - - - S D R
A N L A A I L V H HM D V V S
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V D K
A N L A A F L T - - - - V E T
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V E R
A N L A A F L T - - - - V S R
A N L A A F L T - - - - I T R
A N L A A F L T - - - - V T R
A N L A A F L T - - - - V T R
A N L A A F L T - - - - V T R
A N L A A F L T - - - - V T R
A N L A A F L T - - - - V S R
A N L T A F L T - - - - V S R
A N L A A F L T - - - - V T R
A N L A A F L T - - - - V T R
A S L A A F L T - - - - V A F
A N L A A F L T - - - - V T I
A NM V A V - - - - - - V S R
A NM V T F V S - - - - I K I
A N L A A F L T - - - - V S R
A K L A A F L T - - - - I S R
A N L A A H L T - - - - I G R
A N L A A F L T - - - - I S R
A N L A A F L T - - - - V S S
A N L A A F L T - - - - V R N
A N L A A F L T - - - - I R N
A N L A A F L T - - - - V R N
A N L A A F L T - - - - V S R
A N L A A F L T - - - - V R N
A N L A A F L T - - - - V A R
A N L A A Y L T - - - - A A R
A N L A A F L T - - - - V A R
A N L A A F L T - - - - A A R
A NM A A F L T - - - - T T R
A N L A A F L T - - - - I K R
A N L A A F L G - - - - R A N
A N L A A F L T - - - - V K P
A N L A A F L G - - - - R A N
A N L A A F L T - - - - V K P
A N L A A F L T - - - - K T N
A N L A A F L T - - - - V K P
A N L A A F L T - - - - Q T R
A N L A A F L S - - - - R P S
S N L G A F L T - - - - V E R
S T V A A F L T - - - - V E R
I N L T P F L K - - - - A S K
S N L T P F L M - - - - A S K
F N L T F F I N - - - - S N K
L N L T H F V T - - - - T N K
A N L A A Y F T - - - - G E R
A N L A A F F T - - - - A Q N
2 4 + 8 2 + 7 2 1 2 5
F S F L E P F S Y E V
+ + + 8 + 9 6 - - - - 5 3 5
A N L A A F L T H HM D V E R
553 653 553 653
GluN1_Adi D S F L R P F Q I N L A N L A A F L V - - - - L D R
+
intensity of blue background and by a bar chart at the bottom of the second column. Protein numbering corresponds to mature human GluN2B sequence. Figure was prepared with Jalview v2.10.4b1 (78). Species are identified by a three letters code: Branchiostoma belcheri, Bbe; Branchiostoma floridae, Bfl; Branchiostoma lanceolatum, Bla; Ciona intestinalis, Cin; Ptychodera flava, Pfl; Saccoglossus kowalevskii, Sko; Acanthaster planci, Apl; Strongylocentrotus purpuratus, Spu; Apis mellifera, Ame; Priapulus caudatus, Pca; Strigamia maritima, Sma; Lottia gigantea, Lgi; Capitella teleta, Cte; Nematostella vectensis, Nve; Acropora digitifera, Adi. Gene names are taken from Ramos-Vicente et al. (79).
Table S1. Untargeted analysis of plasma sphingolipid profile in the GRIN2B(P553T) patient before and after L-serine dietary supplementation. Table representing
Mean SD Pre-treatment L-Ser Pre-treatment L-Ser
Ceramide_(34:1)_C34H67O3N 1,46E+06 1,91E+05 1,18E+06 1,50E+06 0,81 1,03
Ceramide_(36:1)_C36H71O3N 7,07E+05 1,77E+05 6,51E+05 7,73E+05 0,92 1,09
Ceramide_(38:1)_C38H75O3N 1,07E+06 1,37E+05 7,80E+05 1,21E+06 0,73 1,13
Ceramide_(39:1)_C39H77O3N 6,57E+05 8,56E+04 2,93E+05 4,05E+05 0,45 0,62
Ceramide_(40:2)_C40H77O3N 1,22E+06 1,20E+05 8,65E+05 1,11E+06 0,71 0,91
Ceramide_(40:1)_C40H79O3N 5,98E+06 7,64E+05 4,66E+06 5,75E+06 0,78 0,96
Ceramide_(41:2)_C41H79O3N 1,37E+06 2,26E+05 7,89E+05 9,31E+05 0,58 0,68
Ceramide_(41:1)_C41H81O3N 7,08E+06 9,76E+05 4,42E+06 5,47E+06 0,62 0,77
Ceramide_(42:2)_C42H81O3N 1,15E+07 2,83E+05 9,44E+06 1,20E+07 0,82 1,04
Ceramide_(42:1)_C42H83O3N 1,85E+07 1,06E+06 1,61E+07 2,00E+07 0,87 1,08
Ceramide_(43:2)_C43H83O3N 1,48E+06 7,07E+03 5,57E+05 7,14E+05 0,38 0,48
Ceramide_(43:1)_C43H85O3N 3,26E+06 3,54E+05 1,54E+06 1,96E+06 0,47 0,60
Ceramide_(44:2)_C44H85O3N 2,49E+05 7,07E+02 2,25E+05 3,03E+05 0,91 1,22
Ceramide_(44:1)_C44H87O3N 8,90E+05 5,87E+04 9,02E+05 1,14E+06 1,01 1,28
Ceramide_(38:0)_C38H77O3N 3,37E+05 1,84E+04 2,36E+05 4,15E+05 0,70 1,23
Ceramide_(40:0)_C40H81O3N 1,90E+06 3,32E+05 1,25E+06 1,32E+06 0,66 0,70
Ceramide_(41:0)_C41H83O3N 1,32E+06 4,65E+05 5,82E+05 6,05E+05 0,44 0,46
Ceramide_(42:0)_C42H85O3N 3,46E+06 1,34E+05 1,20E+06 1,51E+06 0,35 0,44
Ceramide_(43:0)_C43H87O3N 4,37E+05 8,49E+03 3,26E+05 3,20E+05 0,75 0,73
Ceramide_(44:0)_C44H89O3N 4,60E+05 6,01E+04 4,46E+05 6,04E+05 0,97 1,31
Total Ceramides 6,33E+07 1,24E+06 4,64E+07 5,80E+07 0,73 0,92
Sphingomyelin_(32:2)_C37H73N2O6P 8,46E+06 1,21E+06 2,79E+06 3,04E+06 0,33 0,36
Sphingomyelin_(32:1)_C37H75N2O6P 2,68E+08 1,41E+06 1,07E+08 1,19E+08 0,40 0,44
Sphingomyelin_(32:0)_C37H77N2O6P 5,53E+06 3,25E+05 1,40E+06 1,49E+06 0,25 0,27
Sphingomyelin_(33:2)_C38H75N2O6P 3,31E+06 4,60E+05 9,11E+05 1,06E+06 0,28 0,32
Sphingomyelin_(33:1)_C38H77N2O6P 7,13E+07 2,55E+06 2,90E+07 3,31E+07 0,41 0,46
Sphingomyelin_(34:3)_C39H75N2O6P 2,79E+06 3,46E+05 7,34E+05 8,73E+05 0,26 0,31
Sphingomyelin_(34:2)_C39H77N2O6P 1,76E+08 7,78E+06 9,72E+07 1,10E+08 0,55 0,63
Sphingomyelin_(34:1)_C39H79N2O6P 1,43E+09 1,13E+08 9,85E+08 1,12E+09 0,69 0,78
Sphingomyelin_(34:0)_C39H81N2O6P 6,13E+07 2,90E+06 5,77E+07 4,03E+07 0,94 0,66
Sphingomyelin_(35:2)_C40H79N2O6P 5,47E+06 3,89E+05 2,06E+06 2,44E+06 0,38 0,45
Sphingomyelin_(35:1)_C40H81N2O6P 3,87E+07 1,70E+06 1,79E+07 2,15E+07 0,46 0,56
Sphingomyelin_(36:3)_C41H79N2O6P 1,96E+07 2,83E+05 1,06E+07 1,23E+07 0,54 0,63
Sphingomyelin_(36:2)_C41H81N2O6P 1,22E+08 1,48E+07 7,67E+07 8,75E+07 0,63 0,72
Sphingomyelin_(36:1)_C41H83N2O6P 2,62E+08 1,06E+07 1,83E+08 2,01E+08 0,70 0,77
Sphingomyelin_(36:0)_C41H85N2O6P 1,59E+07 2,76E+06 8,81E+06 9,93E+06 0,56 0,63
Sphingomyelin_(37:2)_C42H83N2O6P 4,14E+06 6,93E+05 1,57E+06 1,61E+06 0,38 0,39
Sphingomyelin_(37:1)_C42H85N2O6P 1,94E+07 1,84E+06 8,16E+06 8,72E+06 0,42 0,45
Sphingomyelin_(38:2)_C43H85N2O6P 6,92E+07 7,50E+06 4,75E+07 5,45E+07 0,69 0,79
Sphingomyelin_(38:1)_C43H87N2O6P 1,92E+08 7,07E+06 1,26E+08 1,46E+08 0,66 0,76
Sphingomyelin_(39:2)_C44H87N2O6P 1,18E+07 9,90E+05 3,80E+06 4,15E+06 0,32 0,35
Sphingomyelin_(39:1)_C44H89N2O6P 7,50E+07 1,84E+06 3,00E+07 3,58E+07 0,40 0,48
Sphingomyelin_(40:3)_C45H87N2O6P 2,12E+07 2,33E+06 9,83E+06 1,11E+07 0,46 0,52
Sphingomyelin_(40:2)_C45H89N2O6P 2,89E+08 2,83E+06 1,59E+08 1,83E+08 0,55 0,63
Sphingomyelin_(40:1)_C45H91N2O6P 3,86E+08 1,41E+07 2,51E+08 2,92E+08 0,65 0,76
Sphingomyelin_(41:3)_C46H89N2O6P 1,31E+07 1,63E+06 3,94E+06 4,82E+06 0,30 0,37
Sphingomyelin_(41:2)_C46H91N2O6P 1,54E+08 3,54E+06 6,72E+07 7,98E+07 0,44 0,52
Sphingomyelin_(41:1)_C46H93N2O6P 1,90E+08 9,19E+06 9,83E+07 1,17E+08 0,52 0,62
Sphingomyelin_(42:3)_C47H91N2O6P 3,66E+08 2,47E+07 2,26E+08 2,62E+08 0,62 0,72
Sphingomyelin_(42:2)_C47H93N2O6P 7,93E+08 1,19E+08 5,32E+08 6,25E+08 0,67 0,79
Sphingomyelin_(42:1)_C47H95N2O6P 2,42E+08 5,37E+07 1,73E+08 2,10E+08 0,71 0,87
Sphingomyelin_(43:3)_C48H93N2O6P 9,75E+06 2,62E+06 2,38E+06 2,85E+06 0,24 0,29
Sphingomyelin_(43:2)_C48H95N2O6P 3,40E+07 1,10E+07 1,00E+07 1,23E+07 0,29 0,36
Sphingomyelin_(43:1)_C48H97N2O6P 1,67E+07 4,60E+06 6,21E+06 7,43E+06 0,37 0,45
Sphingomyelin_(44:3)_C49H95N2O6P 4,15E+06 8,34E+05 3,06E+06 4,19E+06 0,74 1,01
Sphingomyelin_(44:2)_C49H97N2O6P 4,16E+06 9,19E+05 3,91E+06 5,02E+06 0,94 1,21
Sphingomyelin_(44:1)_C49H99N2O6P 1,57E+06 4,53E+05 1,05E+06 1,65E+06 0,67 1,05
Total Sphingomyelins 5,38E+09 3,06E+08 3,34E+09 3,83E+09 0,62 0,71
Sphingosine_d18:1_C18H37NO2
Total Sphingosines 5,41E+05 8,20E+04 4,06E+05 6,05E+05 0,75 1,12
Sp
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Controls Patient Fold-change (patient vs. controls)C
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quantitative analysis of untargeted phospholipid profile of control individuals and the GRIN2B(P553T) patient. The values correspond to the chromatographic peak area (arbitrary units). The ratios between the chromatographic peak areas of the sphingolipids detected in the patient (before and after L-serine treatment) and controls are represented in the right column (in bold, phospholipids with a fold-change < 0.5 or >1 .5).
