Table of SynGAP1 Isoform α2 (UniProt Q96PV0-1) Missense Variants.

c.dna	Variant	SGM Consensus	Domain	ClinVar			gnomAD			ESM1b		AlphaMissense			REVEL		FoldX			Rosetta		Foldetta		PremPS		PROVEAN		PolyPhen-2 HumDiv		PolyPhen-2 HumVar		FATHMM		SIFT				PAM		Physical		SASA		Normalized B-factor backbone		Normalized B-factor sidechain		SynGAP Structural Annotation									DOI
				Clinical Status	Review	Subm.	ID	Allele count	Allele freq.	LLR score	Prediction	Pathogenicity	Class	Optimized	Score	Prediction	Average ΔΔG	Prediction	StdDev	ΔΔG	Prediction	ΔΔG	Prediction	ΔΔG	Prediction	Score	Prediction	pph2_prob	Prediction	pph2_prob	Prediction	Nervous System Score	Prediction	Prediction	Status	Conservation	Sequences	PAM250	PAM120	Hydropathy Δ	MW Δ	Average	Δ	Δ	StdDev	Δ	StdDev	Secondary	Tertiary bonds	Inside out	GAP-Ras interface	At membrane	No effect	MD Alert	Verdict	Description
c.1540A>T	I514F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.383	Likely Pathogenic	0.962	Likely Pathogenic	Likely Pathogenic	0.601	Likely Pathogenic	2.35	Destabilizing	0.3	3.74	Destabilizing	3.05	Destabilizing	0.93	Ambiguous	-3.98	Deleterious	0.997	Probably Damaging	0.993	Probably Damaging	2.89	Benign	0.00	Affected	3.37	35	0	1	-1.7	34.02
c.2435C>A	P812H		SH3-binding motif	Uncertain		2	6-33442987-C-A	3	1.86e-6	-7.470	In-Between	0.698	Likely Pathogenic	Likely Benign	0.272	Likely Benign										-2.81	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	2.68	Benign	0.00	Affected	4.32	4	0	-2	-1.6	40.02
c.2912C>A	P971H	Likely Benign		Uncertain		1	6-33443464-C-A	1	6.20e-7	-5.243	Likely Benign	0.086	Likely Benign	Likely Benign	0.039	Likely Benign										-1.11	Neutral	0.898	Possibly Damaging	0.477	Possibly Damaging	3.89	Benign	0.00	Affected	4.32	2	-2	0	-1.6	40.02
c.3251C>A	P1084H	Likely Benign		Uncertain		1	6-33443803-C-A	1	6.31e-7	-4.125	Likely Benign	0.323	Likely Benign	Likely Benign	0.134	Likely Benign										-3.16	Deleterious	0.997	Probably Damaging	0.840	Possibly Damaging	3.96	Benign	0.00	Affected	3.77	5	-2	0	-1.6	40.02
c.3902C>A	P1301H	Likely Benign		Conflicting		2	6-33451776-C-A	5	3.10e-6	-5.756	Likely Benign	0.104	Likely Benign	Likely Benign	0.232	Likely Benign										-1.13	Neutral	0.642	Possibly Damaging	0.378	Benign	2.79	Benign	0.04	Affected	3.77	5	0	-2	-1.6	40.02
c.1354G>T	V452F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.769	Likely Pathogenic	0.975	Likely Pathogenic	Likely Pathogenic	0.511	Likely Pathogenic	9.21	Destabilizing	0.1	0.37	Likely Benign	4.79	Destabilizing	0.61	Ambiguous	-4.94	Deleterious	0.999	Probably Damaging	0.993	Probably Damaging	3.29	Benign	0.00	Affected	3.37	34	-1	-1	-1.4	48.04	249.4	-35.7	0.0	0.0	0.4	0.1							X	Potentially Pathogenic	The iso-propyl side chain of Val452, located in the middle of an α helix (res. Val441-Ser457), packs against hydrophobic residues in the inter-helix space at the intersection of three α helices (e.g., Leu500, His453, Leu465). In the variant simulations, the larger side chain of Phe452 cannot pack against the opposing α helix (res. Leu489-Glu519) as efficiently as valine. Due to space restrictions, the phenol ring adjusts to make room by rotating slightly sideways in the inter-helix space. Besides this small and local shift, no large-scale effects on the protein structure are seen based on the simulations. However, the size difference between the swapped residues could affect the protein folding process.
c.1673A>G	H558R (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.445	Likely Pathogenic	0.554	Ambiguous	Likely Benign	0.587	Likely Pathogenic	-1.14	Ambiguous	0.1	-0.23	Likely Benign	-0.69	Ambiguous	1.03	Destabilizing	-4.94	Deleterious	0.677	Possibly Damaging	0.239	Benign	-1.24	Pathogenic	0.14	Tolerated	3.37	35	0	2	-1.3	19.05
c.194A>G	H65R	Likely Benign		Uncertain		1	6-33425802-A-G	1	6.20e-7	-1.980	Likely Benign	0.967	Likely Pathogenic	Likely Pathogenic	0.073	Likely Benign										-1.60	Neutral	0.462	Possibly Damaging	0.227	Benign	4.19	Benign	0.00	Affected	4.32	1	2	0	-1.3	19.05
c.2852A>G	H951R	Likely Benign		Likely Pathogenic		1				-4.964	Likely Benign	0.125	Likely Benign	Likely Benign	0.185	Likely Benign										-1.08	Neutral	0.048	Benign	0.029	Benign	5.46	Benign	0.24	Tolerated	3.77	5	2	0	-1.3	19.05
c.2888A>G	H963R	Likely Benign		Uncertain		1	6-33443440-A-G	8	4.96e-6	-8.952	Likely Pathogenic	0.169	Likely Benign	Likely Benign	0.081	Likely Benign										-1.28	Neutral	0.001	Benign	0.003	Benign	4.15	Benign	0.24	Tolerated	3.77	5	2	0	-1.3	19.05
c.1516C>T	L506F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.262	Likely Pathogenic	0.883	Likely Pathogenic	Ambiguous	0.464	Likely Benign	4.92	Destabilizing	0.8	5.76	Destabilizing	5.34	Destabilizing	0.91	Ambiguous	-3.98	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.62	Pathogenic	0.01	Affected	3.37	35	0	2	-1.0	34.02
c.169C>T	L57F	Likely Benign		Uncertain		2				-5.096	Likely Benign	0.459	Ambiguous	Likely Benign	0.051	Likely Benign										-0.78	Neutral	0.824	Possibly Damaging	0.879	Possibly Damaging	3.96	Benign	0.00	Affected	4.32	1	2	0	-1.0	34.02
c.1465C>T	L489F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33438497-C-T	1	6.20e-7	-12.066	Likely Pathogenic	0.965	Likely Pathogenic	Likely Pathogenic	0.724	Likely Pathogenic	1.72	Ambiguous	0.5	1.14	Ambiguous	1.43	Ambiguous	0.56	Ambiguous	-3.76	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	-1.51	Pathogenic	0.01	Affected	3.37	35	2	0	-1.0	34.02	246.4	-17.8	0.0	0.0	0.6	0.1						X		Potentially Benign	The iso-butyl side chain of Leu489, located in the α-helix (res. Leu489-Glu519) within an inter-helix space of four helices (res. Ala461-Phe476, res. Val441-Ser457, and res. Met414-Glu436), packs with hydrophobic residues (e.g., Cys432, Ala448, Lys444, Ala493, Val447, Met468) in the inter-helix space. In the variant simulations, the phenyl ring of the Phe489 side chain can also pack favorably in the hydrophobic region. However, due to the size difference, the aromatic side chain of Phe489 tends to reposition to escape the tight region to accommodate the larger side chain, stacking with Lys444. Although no apparent negative changes are observed during the variant simulation, the size difference between the swapped residues could affect the protein folding process.
c.2305C>T	L769F	Likely Benign		Uncertain		1				-5.044	Likely Benign	0.146	Likely Benign	Likely Benign	0.060	Likely Benign										-0.89	Neutral	0.925	Possibly Damaging	0.510	Possibly Damaging	3.94	Benign	0.02	Affected			2	0	-1.0	34.02
c.2105A>G	Q702R (3D Viewer)		GAP	Uncertain		1				-7.894	In-Between	0.348	Ambiguous	Likely Benign	0.294	Likely Benign	-0.31	Likely Benign	0.1	0.63	Ambiguous	0.16	Likely Benign	0.13	Likely Benign	-3.14	Deleterious	0.909	Possibly Damaging	0.889	Possibly Damaging	3.43	Benign	0.02	Affected	3.47	10	1	1	-1.0	28.06	270.3	-52.9	0.0	0.0	0.0	0.1		X						Potentially Pathogenic	The carboxamide side chain of Gln702 is located at the end and outer surface of an α-helix (res. Leu685-Gln702), where it does not directly form hydrogen bonds with any residues in the WT simulations. In the variant simulations, the positively charged guanidinium group of Arg702 forms a salt bridge with the negatively charged carboxylate group of Glu698 on the same helix and/or hydrogen bonds with the backbone carbonyl group of Ala438 on an opposite α-helix (res. Tyr428-Glu436). Consequently, the residue swap could strengthen the tertiary structure assembly, which could have either positive or negative effects on its function.
c.2962C>T	L988F	Likely Benign		Uncertain		1	6-33443514-C-T	1	6.20e-7	-4.368	Likely Benign	0.356	Ambiguous	Likely Benign	0.135	Likely Benign										-1.70	Neutral	0.977	Probably Damaging	0.900	Possibly Damaging	2.69	Benign	0.00	Affected	4.32	2	2	0	-1.0	34.02
c.3125A>G	Q1042R	Likely Benign		Uncertain		2	6-33443677-A-G	2	1.24e-6	-2.928	Likely Benign	0.413	Ambiguous	Likely Benign	0.300	Likely Benign										-1.39	Neutral	0.586	Possibly Damaging	0.120	Benign	5.48	Benign	0.12	Tolerated	3.77	5	1	1	-1.0	28.06
c.3773A>G	Q1258R	Likely Pathogenic	Coiled-coil	Uncertain		1				-10.971	Likely Pathogenic	0.931	Likely Pathogenic	Ambiguous	0.316	Likely Benign										-3.19	Deleterious	0.994	Probably Damaging	0.988	Probably Damaging	2.00	Pathogenic	0.00	Affected			1	1	-1.0	28.06
c.3906G>C	L1302F			Uncertain		1				-5.674	Likely Benign	0.148	Likely Benign	Likely Benign	0.211	Likely Benign										-2.70	Deleterious	0.960	Probably Damaging	0.657	Possibly Damaging	1.53	Pathogenic	0.00	Affected			2	0	-1.0	34.02
c.558G>C	L186F	Likely Pathogenic		Uncertain		1				-11.861	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.132	Likely Benign										-3.03	Deleterious	0.009	Benign	0.012	Benign	3.50	Benign	0.00	Affected			2	0	-1.0	34.02
c.600G>C	L200F (3D Viewer)		PH	Uncertain		1	6-33435242-G-C	2	1.24e-6	-7.606	In-Between	0.592	Likely Pathogenic	Likely Benign	0.094	Likely Benign	1.00	Ambiguous	0.5	1.45	Ambiguous	1.23	Ambiguous	0.43	Likely Benign	-1.97	Neutral	0.997	Probably Damaging	0.916	Probably Damaging	4.02	Benign	0.17	Tolerated	3.46	9	2	0	-1.0	34.02	250.4	-15.1	0.6	0.2	0.5	0.0						X		Uncertain	Leu200, a hydrophobic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another hydrophobic residue, phenylalanine. Both the phenyl group of Phe200 and the branched iso-butyl hydrocarbon sidechain of Leu200 occupy an inward hydrophobic niche (e.g., Leu246, Val222, Phe231) during the simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.1594A>C	T532P (3D Viewer)	Likely Benign	GAP	Benign		1				-2.143	Likely Benign	0.061	Likely Benign	Likely Benign	0.201	Likely Benign	-0.30	Likely Benign	0.2	0.06	Likely Benign	-0.12	Likely Benign	0.08	Likely Benign	-0.90	Neutral	0.005	Benign	0.008	Benign	-1.28	Pathogenic	0.18	Tolerated	3.37	35	0	-1	-0.9	-3.99	174.2	35.1	0.4	0.0	0.1	0.0						X		Potentially Benign	Thr532 is located on an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560) facing the membrane. In the WT simulations, the hydroxyl group of Thr532 occasionally forms hydrogen bonds with the backbone atoms of other loop residues without any specific interaction. In the variant simulations, the Pro532 residue swap does not cause structural changes. Although hydrophilic residues seem more favorable in the loop, the pyrrolidine side chain of proline is well suited for unstructured protein regions such as loops. However, due to its location at the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.
c.2071A>C	T691P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-13.801	Likely Pathogenic	0.905	Likely Pathogenic	Ambiguous	0.214	Likely Benign	5.04	Destabilizing	0.4	6.09	Destabilizing	5.57	Destabilizing	1.27	Destabilizing	-3.43	Deleterious	1.000	Probably Damaging	0.952	Probably Damaging	3.43	Benign	0.06	Tolerated	3.43	14	0	-1	-0.9	-3.99	188.9	33.0	0.1	0.0	-0.6	0.0	X	X						Potentially Pathogenic	The hydroxyl side chain of Thr691, located in an α-helix (res. Leu696-Leu685), can form hydrogen bonds with the backbone carbonyl and the side chain guanidinium group of Arg687. This interaction facilitates the simultaneous formation of salt bridges between Arg687 and Glu688 on the same α-helix. Additionally, Thr691 occasionally interacts with the thioether side chain of Met409 in an anti-parallel β-sheet of the C2 domain (res. Ile411-Ala399), although this interaction is not consistently maintained throughout the WT simulations. In the variant simulations, the pyrrolidine side chain of Pro691 lacks hydrogen bond donors, making a similar setup impossible. Moreover, proline lacks a free amide group necessary for hydrogen bonding with the carbonyl group of Arg687, introducing a slight bend in the α-helix and compromising its integrity.
c.1153T>C	S385P (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438058-T-C			-5.431	Likely Benign	0.123	Likely Benign	Likely Benign	0.385	Likely Benign	0.91	Ambiguous	0.6	-0.90	Ambiguous	0.01	Likely Benign	0.19	Likely Benign	-0.26	Neutral	0.676	Possibly Damaging	0.693	Possibly Damaging	4.63	Benign	0.04	Affected	4.32	3	1	-1	-0.8	10.04	210.3	18.5	1.8	0.9	0.3	0.0								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycine residues, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Pro385 is potentially tolerated in the Ω loop. However, since the effects on Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1558T>C	S520P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.707	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.855	Likely Pathogenic	3.72	Destabilizing	0.8	8.86	Destabilizing	6.29	Destabilizing	0.83	Ambiguous	-4.57	Deleterious	0.997	Probably Damaging	0.986	Probably Damaging	-1.32	Pathogenic	0.01	Affected			1	-1	-0.8	10.04
c.1600T>C	S534P (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438843-T-C	3	1.86e-6	-5.056	Likely Benign	0.265	Likely Benign	Likely Benign	0.203	Likely Benign	-0.40	Likely Benign	0.2	0.35	Likely Benign	-0.03	Likely Benign	0.47	Likely Benign	-3.81	Deleterious	0.993	Probably Damaging	0.993	Probably Damaging	3.32	Benign	0.05	Affected	3.37	35	-1	1	-0.8	10.04
c.2068T>C	S690P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.568	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.431	Likely Benign	4.84	Destabilizing	0.3	4.40	Destabilizing	4.62	Destabilizing	1.42	Destabilizing	-4.77	Deleterious	0.998	Probably Damaging	0.790	Possibly Damaging	3.44	Benign	0.01	Affected	3.42	17	1	-1	-0.8	10.04	207.5	15.1	0.1	0.0	-0.1	0.2	X	X						Potentially Pathogenic	The hydroxyl side chain of Ser690, located in an α-helix (res. Leu696-Leu685), forms a hydrogen bond with the backbone carbonyl group of Ser410 in an anti-parallel β-sheet of the C2 domain (res. Ile411-Ala399). In the variant simulations, the pyrrolidine side chain of Pro690 cannot form hydrogen bonds with the C2 domain residue, resulting in the loss of this inter-domain connection. Additionally, prolines lack a free amide group necessary for hydrogen bonding with the carbonyl group of Gly686, introducing a slight bend in the α-helix and compromising its integrity.
c.2863T>C	S955P	Likely Benign		Uncertain		1	6-33443415-T-C	3	1.86e-6	-2.584	Likely Benign	0.073	Likely Benign	Likely Benign	0.098	Likely Benign										-0.75	Neutral	0.001	Benign	0.004	Benign	2.33	Pathogenic	0.00	Affected	3.77	5	1	-1	-0.8	10.04
c.313T>C	S105P	Likely Benign		Uncertain		1				-3.631	Likely Benign	0.166	Likely Benign	Likely Benign	0.204	Likely Benign										0.03	Neutral	0.808	Possibly Damaging	0.212	Benign	4.00	Benign	0.00	Affected	4.32	1	-1	1	-0.8	10.04
c.3394T>C	S1132P	Likely Benign		Conflicting		3	6-33443946-T-C	1	6.74e-7	-1.423	Likely Benign	0.144	Likely Benign	Likely Benign	0.301	Likely Benign										0.38	Neutral	0.003	Benign	0.006	Benign	5.40	Benign	0.28	Tolerated	4.32	4	1	-1	-0.8	10.04
c.3424T>C	S1142P	Likely Benign		Likely Benign		1	6-33444459-T-C	1	6.20e-7	-2.713	Likely Benign	0.222	Likely Benign	Likely Benign	0.107	Likely Benign										-2.19	Neutral	0.918	Possibly Damaging	0.761	Possibly Damaging	2.64	Benign	0.00	Affected	4.32	4	-1	1	-0.8	10.04
c.82T>C	S28P	Likely Benign		Uncertain		1				-3.309	Likely Benign	0.051	Likely Benign	Likely Benign	0.047	Likely Benign										1.37	Neutral	0.000	Benign	0.000	Benign	4.53	Benign	0.00	Affected	4.32	1	1	-1	-0.8	10.04
c.703T>C	S235P (3D Viewer)	Likely Pathogenic	PH	Likely Pathogenic		1				-14.857	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.870	Likely Pathogenic	4.02	Destabilizing	0.1	6.91	Destabilizing	5.47	Destabilizing	1.23	Destabilizing	-4.24	Deleterious	0.917	Possibly Damaging	0.446	Benign	5.47	Benign	0.01	Affected	3.40	14	1	-1	-0.8	10.04	201.5	17.0	0.1	0.0	-0.6	0.0		X						Potentially Pathogenic	In the WT, the hydroxyl group of Ser235, located in a β-α loop between an anti-parallel β sheet strand (res. Gly227-Phe231) and an α helix (residues Ala236-Val250), forms hydrogen bonds with the GAP domain loop residue Glu680 and with the backbone amide groups of Ala237 and Glu238 from the α helix. In the variant simulations, the pyrrolidine ring of Pro235 cannot stabilize the α helix end or maintain tertiary bonding interactions between the PH and GAP domains via hydrogen bonding as effectively as serine.
c.1511A>G	K504R (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438543-A-G	2	1.24e-6	-4.365	Likely Benign	0.088	Likely Benign	Likely Benign	0.238	Likely Benign	0.13	Likely Benign	0.1	0.51	Ambiguous	0.32	Likely Benign	0.94	Ambiguous	-2.16	Neutral	0.002	Benign	0.015	Benign	-1.41	Pathogenic	0.11	Tolerated	3.37	35	2	3	-0.6	28.01
c.1913A>G	K638R (3D Viewer)	Likely Benign	GAP	Uncertain		1				-2.700	Likely Benign	0.110	Likely Benign	Likely Benign	0.216	Likely Benign	0.09	Likely Benign	0.1	-0.04	Likely Benign	0.03	Likely Benign	0.53	Ambiguous	-2.55	Deleterious	0.649	Possibly Damaging	0.240	Benign	3.41	Benign	0.13	Tolerated	3.37	31	2	3	-0.6	28.01
c.2408A>G	K803R	Likely Benign	SH3-binding motif	Uncertain		1				-2.281	Likely Benign	0.097	Likely Benign	Likely Benign	0.018	Likely Benign										-1.52	Neutral	0.103	Benign	0.038	Benign	2.38	Pathogenic	0.00	Affected	3.77	5	3	2	-0.6	28.01
c.323A>G	K108R	Likely Benign		Uncertain		1	6-33432188-A-G	6	3.72e-6	-2.892	Likely Benign	0.148	Likely Benign	Likely Benign	0.184	Likely Benign										0.37	Neutral	0.993	Probably Damaging	0.956	Probably Damaging	4.22	Benign	1.00	Tolerated	3.61	5	3	2	-0.6	28.01
c.1147G>T	G383W (3D Viewer)		C2	Uncertain		1	6-33438052-G-T	1	6.22e-7	-10.161	Likely Pathogenic	0.439	Ambiguous	Likely Benign	0.469	Likely Benign	5.81	Destabilizing	3.6	4.44	Destabilizing	5.13	Destabilizing	0.08	Likely Benign	-1.01	Neutral	0.959	Probably Damaging	0.704	Possibly Damaging	4.09	Benign	0.00	Affected	4.32	7	-2	-7	-0.5	129.16
c.1121C>A	S374Y (3D Viewer)		C2	Uncertain		1				-7.774	In-Between	0.344	Ambiguous	Likely Benign	0.310	Likely Benign	0.71	Ambiguous	1.2	0.66	Ambiguous	0.69	Ambiguous	-0.02	Likely Benign	-1.18	Neutral	0.875	Possibly Damaging	0.271	Benign	5.41	Benign	0.01	Affected	4.32	13	-3	-2	-0.5	76.10	237.3	-76.9	0.5	0.4	0.5	0.3								Uncertain	Ser374 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus, large and relatively hydrophobic residues like tyrosine are rarely tolerated. Additionally, the hydroxyl group of Tyr374 frequently forms various hydrogen bonds with other loop residues in the variant simulations. Although no negative structural effects are observed in the variant simulations, Tyr374 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.2525C>A	S842Y	Likely Pathogenic		Likely Pathogenic		1				-16.124	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.191	Likely Benign										-4.28	Deleterious	0.944	Possibly Damaging	0.676	Possibly Damaging	1.97	Pathogenic	0.00	Affected	3.77	5	-3	-2	-0.5	76.10
c.3395C>A	S1132Y	Likely Benign		Likely Benign		1				-5.894	Likely Benign	0.392	Ambiguous	Likely Benign	0.401	Likely Benign										-1.76	Neutral	0.500	Possibly Damaging	0.208	Benign	5.40	Benign	0.09	Tolerated	4.32	4	-3	-2	-0.5	76.10
c.3413C>A	S1138Y			Uncertain		1	6-33444448-C-A	3	1.86e-6	-6.610	Likely Benign	0.449	Ambiguous	Likely Benign	0.391	Likely Benign										-2.51	Deleterious	0.997	Probably Damaging	0.996	Probably Damaging	5.41	Benign	0.05	Affected	4.32	4	-2	-3	-0.5	76.10
c.1030G>A	G344S (3D Viewer)	Likely Pathogenic	C2	Pathogenic		5				-11.254	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.790	Likely Pathogenic	9.02	Destabilizing	0.7	6.08	Destabilizing	7.55	Destabilizing	0.98	Ambiguous	-5.28	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-0.45	Pathogenic	0.04	Affected	3.37	25	1	0	-0.4	30.03	217.3	-51.7	0.0	0.1	0.2	0.1			X				X	Potentially Pathogenic	Because Gly344 lacks a proper side chain, it allows the anti-parallel β sheet strand (res. Gly341-Pro349) to have a slight twist. Within a β strand, side chains normally alternate between outward and inward positions, but glycine is an exception as it allows the alternating pattern to skip a residue. Introducing serine or any other residue with a side chain at position 344 prevents this unique skip in the alternating pattern, causing structural strain or likely preventing correct folding altogether. Additionally, Tyr342 shields Gly344 from the solvent, contributing to twist formation in the β sheet and stabilizing the β-strand.In the variant simulations, the side chain of Ser344 assumes the inward position. However, the hydrophobic niche formed by multiple C2 domain residues (e.g., Val365, Val343, Leu327) is not accommodating for its hydroxyl group. The outward position, not seen in the simulations, would be equally disadvantageous due to the presence of hydrophobic residues on that side as well (e.g., Leu345, Tyr342). Serine is also not well-suited for twist formation, as it tends to suppress twisting and bending in β sheets. At this position, the hydroxyl group of Ser344 could also form hydrogen bonds with the backbone atoms of the Gly-rich Ω loop in the C2 domain (e.g., Thr366, Leu367, Gly378; res. Pro364-Pro398), potentially adversely affecting membrane-loop dynamics and ultimately compromising the stability of the SynGAP-membrane association.
c.1108G>A	G370S (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438013-G-A	15	9.31e-6	-3.533	Likely Benign	0.081	Likely Benign	Likely Benign	0.282	Likely Benign	2.83	Destabilizing	2.0	1.05	Ambiguous	1.94	Ambiguous	-0.02	Likely Benign	0.47	Neutral	0.000	Benign	0.000	Benign	1.33	Pathogenic	0.77	Tolerated	3.42	19	1	0	-0.4	30.03	196.6	-49.6	0.9	2.2	-0.1	0.4								Uncertain	Gly370 is located in the Gly-rich Ω loop (res. Pro364- Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because, the Ω loop is assumed to be directly interacting with the membrane, it is only seen to move arbitrarily throughout the WT solvent simulations. The Ω loop is potentially playing a crucial loop in the SynGAP-membrane complex association, stability and dynamics, regardless, this aspect cannot be addressed through the solvent simulations only. The Ω-loops are known to have a major role in protein functions that requires flexibility and thus, they are rich in glycines, prolines and to a lesser extent, hydrophilic residues to ensure maximum flexibility. Thus, Ser370 in the variant is potentially tolerated in the Ω loop. However, since the effect on the Gly-rich Ω loop dynamics can only be well-studied through the SynGAP-membrane complex, no definite conclusions can be withdrawn.
c.127G>A	G43S	Likely Benign		Uncertain		2	6-33423536-G-A	1	6.20e-7	-3.301	Likely Benign	0.078	Likely Benign	Likely Benign	0.057	Likely Benign										-0.30	Neutral	0.162	Benign	0.096	Benign	4.29	Benign	0.00	Affected	4.32	1	1	0	-0.4	30.03
c.1339G>C	V447L (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.136	Likely Benign	0.491	Ambiguous	Likely Benign	0.180	Likely Benign	-1.13	Ambiguous	0.1	0.54	Ambiguous	-0.30	Likely Benign	0.03	Likely Benign	-0.29	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.61	Benign	0.90	Tolerated	3.37	32	1	2	-0.4	14.03
c.1150G>A	G384S (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438055-G-A	1	6.22e-7	-5.243	Likely Benign	0.090	Likely Benign	Likely Benign	0.315	Likely Benign	1.92	Ambiguous	0.2	1.66	Ambiguous	1.79	Ambiguous	0.19	Likely Benign	-0.67	Neutral	0.980	Probably Damaging	0.968	Probably Damaging	1.33	Pathogenic	0.04	Affected	4.32	2	1	0	-0.4	30.03	202.4	-49.8	0.5	1.0	-0.2	0.0								Uncertain	Gly384 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycines, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Ser384 is potentially tolerated in the Ω loop, although the hydroxyl group of Ser384 forms various hydrogen bonds with several other loop residues in the variant simulations. However, since the effects on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1198G>C	V400L (3D Viewer)	Likely Benign	C2	Benign		1	6-33438103-G-C	22	1.36e-5	-1.000	Likely Benign	0.137	Likely Benign	Likely Benign	0.325	Likely Benign	-0.71	Ambiguous	0.2	0.39	Likely Benign	-0.16	Likely Benign	-0.29	Likely Benign	-0.60	Neutral	0.001	Benign	0.001	Benign	5.33	Benign	0.64	Tolerated	3.38	27	2	1	-0.4	14.03	251.0	-30.1	0.0	0.0	0.7	0.1						X		Potentially Benign	The iso-propyl side chain of Val400, located in an anti-parallel β sheet strand (res. Ala399-Ile411), hydrophobically packs against hydrophobic residues within the anti-parallel β sheet of the C2 domain (e.g., Ile268, Ala404, Leu325, Leu402). Val400 is swapped for another hydrophobic residue, leucine, whose branched hydrocarbon side chain is of a comparable size and thus packs favorably within the C2 domain. In short, the residue swap has no apparent negative effect on the structure based on the variant simulations.	10.1016/j.ajhg.2020.11.011
c.1483G>A	E495K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.478	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.869	Likely Pathogenic	0.15	Likely Benign	0.2	0.66	Ambiguous	0.41	Likely Benign	0.70	Ambiguous	-3.91	Deleterious	0.999	Probably Damaging	0.994	Probably Damaging	-1.29	Pathogenic	0.01	Affected	3.37	35	1	0	-0.4	-0.94
c.1306G>A	E436K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.869	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.829	Likely Pathogenic	0.56	Ambiguous	0.1	2.86	Destabilizing	1.71	Ambiguous	0.82	Ambiguous	-3.77	Deleterious	0.994	Probably Damaging	0.951	Probably Damaging	4.71	Benign	0.02	Affected	3.37	29	0	1	-0.4	-0.94	186.8	39.8	0.0	0.0	-0.2	0.0	X	X		X				Potentially Pathogenic	The carboxylate group of Glu436, located on the α helix (res. Met414-Glu436), forms a salt bridge with the amino group of the Lys444 side chain on an opposing α helix (res. Val441-Ser457). The backbone carbonyl of Glu436 also H-bonds with the Lys444 side chain, which helps keep the ends of the two α helices tightly connected. In contrast, in the variant simulations, the salt bridge formation with Lys444 is not possible. Instead, the repelled Lys436 side chain rotates outward, causing a change in the α helix backbone H-bonding: the amide group of Lys444 H-bonds with the carbonyl of Ala433 instead of the carbonyl of Cys432.
c.163C>A	Q55K	Likely Benign		Uncertain		2	6-33423572-C-A	24	1.49e-5	-5.840	Likely Benign	0.612	Likely Pathogenic	Likely Benign	0.085	Likely Benign										-1.21	Neutral	0.140	Benign	0.184	Benign	3.91	Benign	0.00	Affected	4.32	1	1	1	-0.4	0.04
c.1702G>T	V568L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.503	Likely Pathogenic	0.921	Likely Pathogenic	Ambiguous	0.651	Likely Pathogenic	-0.30	Likely Benign	0.3	0.57	Ambiguous	0.14	Likely Benign	0.56	Ambiguous	-2.69	Deleterious	0.511	Possibly Damaging	0.147	Benign	-1.23	Pathogenic	0.04	Affected	3.37	35	1	2	-0.4	14.03
c.1738G>A	G580S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440790-G-A	1	6.20e-7	-10.788	Likely Pathogenic	0.861	Likely Pathogenic	Ambiguous	0.644	Likely Pathogenic	2.84	Destabilizing	0.2	0.59	Ambiguous	1.72	Ambiguous	0.87	Ambiguous	-5.73	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.23	Pathogenic	0.07	Tolerated	3.37	34	1	0	-0.4	30.03	233.9	-49.3	0.8	0.0	0.6	0.1						X		Potentially Benign	Gly580 is located on the outer surface in a short α-α loop turn connecting two α-helices (res. Arg563-Glu578, res. Glu582-Phe608) in the WT simulations. In the variant simulations, the side chain of Ser580 faces outward, and its hydroxyl group does not make any new or additional interactions compared to Gly580 in the WT simulations that could affect the protein structure.
c.1456G>A	E486K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.545	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.435	Likely Benign	0.06	Likely Benign	0.1	0.37	Likely Benign	0.22	Likely Benign	0.41	Likely Benign	-3.58	Deleterious	1.000	Probably Damaging	0.988	Probably Damaging	3.40	Benign	0.12	Tolerated	3.37	35	0	1	-0.4	-0.94	206.8	52.1	-0.3	0.1	0.2	0.0	X			X				Uncertain	Glu486 is located in an α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. It is adjacent to the arginine finger (Arg485) and is expected to closely interact with Ras. The residue swap could affect complex formation with the GTPase and its activation. In the WT simulations, the carboxylate group of Glu486 forms salt bridges with Arg485 and Arg475 on the preceding α-helix (res. Ala461-Phe476). In the variant simulations, Lys486 does not form any specific interactions. Although the amino group of the Lys486 side chain cannot form these salt bridges, no negative effects on the protein structure are observed. Nevertheless, the potential role of Glu486 in SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations, and no definite conclusions can be drawn.
c.187G>A	E63K	Likely Benign		Uncertain		1				-4.976	Likely Benign	0.894	Likely Pathogenic	Ambiguous	0.103	Likely Benign										-0.70	Neutral	0.458	Possibly Damaging	0.678	Possibly Damaging	3.98	Benign	0.00	Affected	4.32	1	1	0	-0.4	-0.94
c.2239G>C	V747L	Likely Benign		Uncertain		1	6-33441704-G-C	2	1.24e-6	-2.790	Likely Benign	0.096	Likely Benign	Likely Benign	0.047	Likely Benign										-0.52	Neutral	0.065	Benign	0.033	Benign	2.67	Benign	0.00	Affected	4.32	2	2	1	-0.4	14.03
c.223G>A	E75K	Likely Benign		Benign/Likely benign		2				-4.020	Likely Benign	0.358	Ambiguous	Likely Benign	0.134	Likely Benign										-1.12	Neutral	0.748	Possibly Damaging	0.017	Benign	4.07	Benign	0.00	Affected			0	1	-0.4	-0.94
c.2401G>A	G801S	Likely Benign	SH3-binding motif	Uncertain		1				-3.665	Likely Benign	0.087	Likely Benign	Likely Benign	0.039	Likely Benign										-0.41	Neutral	0.009	Benign	0.019	Benign	2.76	Benign	0.48	Tolerated	4.32	2	0	1	-0.4	30.03
c.2485G>A	E829K	Likely Pathogenic		Pathogenic		1				-7.527	In-Between	0.807	Likely Pathogenic	Ambiguous	0.194	Likely Benign										-2.65	Deleterious	0.994	Probably Damaging	0.900	Possibly Damaging	2.27	Pathogenic	0.00	Affected	3.77	5	0	1	-0.4	-0.94
c.2514C>A	N838K	Likely Pathogenic		Uncertain		2				-8.470	Likely Pathogenic	0.862	Likely Pathogenic	Ambiguous	0.097	Likely Benign										-2.78	Deleterious	0.997	Probably Damaging	0.995	Probably Damaging	2.69	Benign	0.16	Tolerated	3.77	5	1	0	-0.4	14.07
c.2116G>A	E706K (3D Viewer)		GAP	Uncertain		1				-10.519	Likely Pathogenic	0.833	Likely Pathogenic	Ambiguous	0.080	Likely Benign	1.17	Ambiguous	0.1	0.51	Ambiguous	0.84	Ambiguous	0.08	Likely Benign	-1.51	Neutral	0.345	Benign	0.028	Benign	4.15	Benign	0.52	Tolerated	3.47	10	0	1	-0.4	-0.94	187.1	49.2	0.0	0.0	0.4	0.1						X		Uncertain	The carboxylate side chain of Glu706, located at the end and outer surface of an α-helix (res. Thr704-Gly712), forms a salt bridge with Lys710 and a hydrogen bond with its own backbone amino group at the helix end in the WT simulations. Although Lys706 is unable to make these transient interactions in the variant simulations, there is no apparent negative effect on the protein structure due to the residue swap. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2596G>T	V866L	Likely Benign		Uncertain		1	6-33443148-G-T	1	6.20e-7	-3.352	Likely Benign	0.148	Likely Benign	Likely Benign	0.046	Likely Benign										-0.97	Neutral	0.217	Benign	0.229	Benign	2.71	Benign	0.21	Tolerated	3.82	4	2	1	-0.4	14.03
c.2743G>A	G915S	Likely Benign		Benign		1	6-33443295-G-A	9	5.58e-6	-3.557	Likely Benign	0.083	Likely Benign	Likely Benign	0.050	Likely Benign										-0.88	Neutral	0.801	Possibly Damaging	0.201	Benign	2.73	Benign	0.31	Tolerated	3.77	5	1	0	-0.4	30.03
c.2818G>A	G940S	Likely Benign		Uncertain		1	6-33443370-G-A	1	6.20e-7	-5.451	Likely Benign	0.084	Likely Benign	Likely Benign	0.135	Likely Benign										0.45	Neutral	0.409	Benign	0.253	Benign	2.77	Benign	0.44	Tolerated	3.77	5	1	0	-0.4	30.03
c.2830G>A	G944S	Likely Benign		Benign		1	6-33443382-G-A	13	8.05e-6	-5.303	Likely Benign	0.082	Likely Benign	Likely Benign	0.223	Likely Benign										-0.75	Neutral	0.007	Benign	0.004	Benign	3.77	Benign	0.00	Affected	4.32	4	1	0	-0.4	30.03
c.2845G>A	G949S	Likely Benign		Benign/Likely benign		4	6-33443397-G-A	122	7.56e-5	-5.693	Likely Benign	0.072	Likely Benign	Likely Benign	0.321	Likely Benign										0.30	Neutral	0.611	Possibly Damaging	0.102	Benign	2.23	Pathogenic	0.00	Affected	4.32	4	1	0	-0.4	30.03																10.1016/j.ajhg.2020.11.011
c.2854G>A	G952S	Likely Benign		Conflicting		2	6-33443406-G-A	2	1.24e-6	-6.190	Likely Benign	0.077	Likely Benign	Likely Benign	0.167	Likely Benign										0.19	Neutral	0.000	Benign	0.002	Benign	3.31	Benign	0.07	Tolerated	3.77	5	1	0	-0.4	30.03
c.286G>A	G96S	Likely Benign		Uncertain		1	6-33425894-G-A	5	3.10e-6	-3.049	Likely Benign	0.065	Likely Benign	Likely Benign	0.071	Likely Benign										-0.76	Neutral	0.364	Benign	0.008	Benign	4.25	Benign	0.00	Affected	4.32	1	1	0	-0.4	30.03
c.3160G>A	G1054S	Likely Benign		Benign		1	6-33443712-G-A	32	1.99e-5	-5.294	Likely Benign	0.075	Likely Benign	Likely Benign	0.160	Likely Benign										0.21	Neutral	0.121	Benign	0.013	Benign	4.04	Benign	0.63	Tolerated	3.77	5	1	0	-0.4	30.03
c.3172G>A	G1058S	Likely Benign		Conflicting		3	6-33443724-G-A	114	7.08e-5	-5.178	Likely Benign	0.081	Likely Benign	Likely Benign	0.108	Likely Benign										0.26	Neutral	0.001	Benign	0.001	Benign	5.38	Benign	0.04	Affected	3.77	5	1	0	-0.4	30.03
c.3178G>A	G1060S	Likely Benign		Uncertain		1	6-33443730-G-A			-4.759	Likely Benign	0.082	Likely Benign	Likely Benign	0.376	Likely Benign										-0.08	Neutral	0.271	Benign	0.054	Benign	2.69	Benign	0.49	Tolerated	4.32	2	1	0	-0.4	30.03
c.3181G>A	G1061S	Likely Benign		Uncertain		1				-4.891	Likely Benign	0.079	Likely Benign	Likely Benign	0.283	Likely Benign										-0.68	Neutral	0.004	Benign	0.004	Benign	4.00	Benign	0.00	Affected			1	0	-0.4	30.03
c.3223C>A	Q1075K	Likely Benign		Uncertain		1				-5.135	Likely Benign	0.728	Likely Pathogenic	Likely Benign	0.134	Likely Benign										-0.67	Neutral	0.963	Probably Damaging	0.959	Probably Damaging	2.75	Benign	1.00	Tolerated	3.77	5	1	1	-0.4	0.04
c.3364G>A	G1122S	Likely Benign		Benign/Likely benign		2	6-33443916-G-A	27	1.79e-5	-4.880	Likely Benign	0.072	Likely Benign	Likely Benign	0.189	Likely Benign										-0.08	Neutral	0.022	Benign	0.006	Benign	4.89	Benign	0.92	Tolerated	3.77	5	1	0	-0.4	30.03
c.3520G>A	E1174K	Likely Benign	Coiled-coil	Uncertain		1	6-33444555-G-A	2	1.24e-6	-4.345	Likely Benign	0.898	Likely Pathogenic	Ambiguous	0.442	Likely Benign										-1.59	Neutral	0.962	Probably Damaging	0.367	Benign	5.52	Benign	0.03	Affected	4.32	2	0	1	-0.4	-0.94
c.3529G>A	E1177K	Likely Benign	Coiled-coil	Uncertain		1				-3.413	Likely Benign	0.944	Likely Pathogenic	Ambiguous	0.560	Likely Pathogenic										-1.75	Neutral	0.905	Possibly Damaging	0.637	Possibly Damaging	5.44	Benign	0.11	Tolerated	4.32	2	0	1	-0.4	-0.94
c.3595G>A	E1199K		Coiled-coil	Uncertain		1	6-33446587-G-A	1	6.20e-7	-10.853	Likely Pathogenic	0.954	Likely Pathogenic	Ambiguous	0.171	Likely Benign										-2.26	Neutral	1.000	Probably Damaging	0.995	Probably Damaging	2.52	Benign	0.00	Affected	3.77	5	0	1	-0.4	-0.94
c.3907G>A	G1303S	Likely Benign		Uncertain		1				-2.271	Likely Benign	0.125	Likely Benign	Likely Benign	0.155	Likely Benign										-0.19	Neutral	0.649	Possibly Damaging	0.433	Benign	2.84	Benign	0.18	Tolerated			1	0	-0.4	30.03
c.3949G>A	G1317S	Likely Benign		Conflicting		3	6-33451823-G-A	1	6.26e-7	-3.522	Likely Benign	0.145	Likely Benign	Likely Benign	0.092	Likely Benign										-2.45	Neutral	0.127	Benign	0.045	Benign	4.08	Benign	0.00	Affected	3.77	5	1	0	-0.4	30.03
c.4003G>A	G1335S	Likely Pathogenic		Conflicting		2	6-33451877-G-A	3	2.37e-6	-4.495	Likely Benign	0.986	Likely Pathogenic	Likely Pathogenic	0.362	Likely Benign										-3.79	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	2.04	Pathogenic	0.00	Affected	3.77	5	1	0	-0.4	30.03
c.4006G>A	E1336K	Likely Benign		Benign		2	6-33451880-G-A	6	4.20e-6	-4.697	Likely Benign	0.977	Likely Pathogenic	Likely Pathogenic	0.272	Likely Benign										-2.44	Neutral	0.748	Possibly Damaging	0.079	Benign	3.23	Benign	0.00	Affected	3.77	5	0	1	-0.4	-0.94
c.597C>A	N199K (3D Viewer)		PH	Uncertain		1				-8.198	Likely Pathogenic	0.686	Likely Pathogenic	Likely Benign	0.024	Likely Benign	-0.19	Likely Benign	0.1	0.03	Likely Benign	-0.08	Likely Benign	0.33	Likely Benign	-1.48	Neutral	0.276	Benign	0.083	Benign	4.27	Benign	0.13	Tolerated	3.47	9	1	0	-0.4	14.07	207.8	21.5	-0.1	1.5	0.1	0.0						X		Uncertain	Asn199, located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by a positively charged lysine. On the protein surface, both the carboxamide group of Asn199 and the amino group of Lys199 side chains can form hydrogen bonds with the backbone carbonyl groups of residues (e.g., Ala249) at the end of an α helix (res. Ala236-Lys251). However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.937G>A	E313K (3D Viewer)	Likely Pathogenic	C2	Likely Benign		1				-12.902	Likely Pathogenic	0.959	Likely Pathogenic	Likely Pathogenic	0.575	Likely Pathogenic	0.64	Ambiguous	0.6	1.40	Ambiguous	1.02	Ambiguous	0.75	Ambiguous	-3.31	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	1.90	Pathogenic	0.02	Affected			0	1	-0.4	-0.94
c.958G>C	V320L (3D Viewer)		C2	Uncertain		1	6-33437863-G-C	6	3.72e-6	-6.207	Likely Benign	0.362	Ambiguous	Likely Benign	0.096	Likely Benign	-0.26	Likely Benign	0.2	1.33	Ambiguous	0.54	Ambiguous	0.51	Ambiguous	-1.02	Neutral	0.900	Possibly Damaging	0.373	Benign	1.78	Pathogenic	0.92	Tolerated	3.38	23	2	1	-0.4	14.03	245.8	-10.2	0.3	0.9	0.1	0.3						X		Potentially Benign	The isopropyl side chain of Val310, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Thr305-Asn315, res. Ala322-Asp330), hydrophobically packs with the side chains of nearby residues (e.g., Leu286, Val350, Pro318). The hydrophobic Leu320 side chain mostly forms the same interactions; hence, the residue swap does not seem to negatively affect the protein structure based on the variant simulations.
c.928G>A	E310K (3D Viewer)	Likely Pathogenic	C2	Conflicting		4				-14.601	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.764	Likely Pathogenic	1.97	Ambiguous	1.2	3.66	Destabilizing	2.82	Destabilizing	1.02	Destabilizing	-3.68	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	1.19	Pathogenic	0.01	Affected	3.38	19	0	1	-0.4	-0.94	213.4	58.0	0.1	0.0	0.2	0.1		X						Potentially Pathogenic	The carboxylate group of Glu310, located in an anti-parallel β sheet strand (res. Thr305-Asn315), is ideally positioned to interact with the side chain hydroxyl and backbone amide groups of Thr295 on a twisted anti-parallel β strand (res. Met289-Arg299). Because the carboxylate group can also interact with the GAP domain residues (e.g., Gln612, Tyr614), Glu310 plays a key role in maintaining the tertiary assembly between the C2 and GAP domains. In the variant simulations, the amino group of the Lys310 side chain hydrogen bonds with the GAP domain residues and forms a salt bridge with Glu613. Although no apparent negative effects are seen due to the residue swap, it is possible that the loss of hydrogen bonding with the hydroxyl group of the Thr295 side chain causes problems during folding, potentially compromising the twisting of the β sheet.
c.1231A>G	I411V (3D Viewer)	Likely Benign	GAP	Likely Benign		1				-6.290	Likely Benign	0.385	Ambiguous	Likely Benign	0.212	Likely Benign	0.74	Ambiguous	0.0	0.82	Ambiguous	0.78	Ambiguous	0.99	Ambiguous	-0.86	Neutral	0.935	Possibly Damaging	0.858	Possibly Damaging	3.90	Benign	0.27	Tolerated	3.38	28	4	3	-0.3	-14.03	233.3	28.2	-0.2	0.0	-0.2	0.0						X		Potentially Benign	The sec-butyl side chain of Ile411, located in the hydrophobic space between an anti-parallel β sheet strand (res. Pro398-Ile411) and an α helix (res. Asp684-Gln702), packs against multiple residues (e.g., Met409, Arg259). In the variant simulations, the side chain of Val411 is able to favorably fill the same hydrophobic niche despite its slightly smaller size. In short, the residue swap has no apparent negative effect on the structure based on the simulations.
c.1447A>G	I483V (3D Viewer)		GAP	Conflicting		2				-10.121	Likely Pathogenic	0.523	Ambiguous	Likely Benign	0.228	Likely Benign	1.00	Ambiguous	0.0	0.27	Likely Benign	0.64	Ambiguous	1.02	Destabilizing	-0.86	Neutral	0.914	Possibly Damaging	0.921	Probably Damaging	3.23	Benign	0.03	Affected	3.37	32	3	4	-0.3	-14.03
c.1480A>G	I494V (3D Viewer)		GAP	Conflicting		2	6-33438512-A-G	36	2.23e-5	-7.102	In-Between	0.112	Likely Benign	Likely Benign	0.439	Likely Benign	1.16	Ambiguous	0.0	0.71	Ambiguous	0.94	Ambiguous	1.02	Destabilizing	-0.83	Neutral	0.278	Benign	0.179	Benign	-1.30	Pathogenic	0.07	Tolerated	3.37	35	4	3	-0.3	-14.03	248.6	29.3	0.0	0.0	-1.1	0.5						X		Potentially Benign	The sec-butyl side chain of Ile494, located in an α-helix (res. Leu489-Glu519), packs against hydrophobic residues (e.g., Phe484, Leu465, Trp572, Ala493, Met468) in an inter-helix space (res. Leu489-Glu519 and res. Ala461-Phe476). In the variant simulations, the hydrophobic iso-propyl side chain of Val494, which is of a similar size and has similar physicochemical properties to Ile494 in the WT, resides similarly in the inter-helix hydrophobic space. Thus, no negative effects on the protein structure are observed.
c.2299A>G	I767V	Likely Benign		Uncertain		1				-2.791	Likely Benign	0.064	Likely Benign	Likely Benign	0.096	Likely Benign										0.10	Neutral	0.072	Benign	0.029	Benign	4.21	Benign	1.00	Tolerated	3.64	6	4	3	-0.3	-14.03
c.1888A>G	I630V (3D Viewer)		GAP	Benign/Likely benign		4	6-33440940-A-G	59	3.66e-5	-7.264	In-Between	0.145	Likely Benign	Likely Benign	0.143	Likely Benign	1.33	Ambiguous	0.0	0.94	Ambiguous	1.14	Ambiguous	0.64	Ambiguous	-0.38	Neutral	0.018	Benign	0.011	Benign	-1.37	Pathogenic	0.35	Tolerated	3.37	34	4	3	-0.3	-14.03	235.0	26.2	-0.1	0.0	-0.3	0.1						X		Potentially Benign	The sec-butyl side chain of Ile630, located in an α helix (res. Glu617-Asn635), packs with hydrophobic residues (e.g., Phe594, Leu633, Ile626, Ile602) in the hydrophobic inter-helix space between two α helices (res. Glu617-Asn635 and res. Glu582-Met603).In the variant simulations, the iso-propyl side chain of Val630, which shares a similar size and physicochemical properties with Ile630 in the WT, maintains similar interactions in the inter-helix space. Although no negative structural effects are observed during the simulations, the implications of the residue swap on the complex formation with the GTPase, due to its location, cannot be investigated using solvent-only simulations.
c.2047A>G	I683V (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441306-A-G	2	1.24e-6	-7.588	In-Between	0.138	Likely Benign	Likely Benign	0.112	Likely Benign	0.90	Ambiguous	0.0	0.60	Ambiguous	0.75	Ambiguous	0.76	Ambiguous	-0.78	Neutral	0.538	Possibly Damaging	0.080	Benign	3.35	Benign	0.14	Tolerated	3.42	17	4	3	-0.3	-14.03	215.6	29.1	0.0	0.0	-0.7	0.1						X		Potentially Benign	The sec-butyl side chain of Ile683, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is sterically packed against His453 and Glu688. In the variant simulations, the iso-propyl side chain of Val683 has similar size and physicochemical properties as Ile630 in the WT, and thus, it is able to maintain similar interactions in the inter-helix space. Consequently, no negative structural effects are observed during the simulations due to the residue swap.
c.2695A>G	I899V	Likely Benign		Benign		1	6-33443247-A-G	6	3.72e-6	-2.569	Likely Benign	0.074	Likely Benign	Likely Benign	0.040	Likely Benign										0.09	Neutral	0.220	Benign	0.078	Benign	2.75	Benign	0.92	Tolerated	4.32	4	4	3	-0.3	-14.03
c.2835T>A	H945Q	Likely Benign		Conflicting		2	6-33443387-T-A	3	1.86e-6	-5.248	Likely Benign	0.091	Likely Benign	Likely Benign	0.343	Likely Benign										-0.36	Neutral	0.995	Probably Damaging	0.939	Probably Damaging	5.03	Benign	0.06	Tolerated	4.32	4	3	0	-0.3	-9.01
c.2998A>G	I1000V	Likely Benign		Uncertain		2				-4.102	Likely Benign	0.098	Likely Benign	Likely Benign	0.086	Likely Benign										-0.20	Neutral	0.437	Benign	0.170	Benign	2.76	Benign	0.81	Tolerated	4.32	4	3	4	-0.3	-14.03
c.303C>A	H101Q	Likely Benign		Uncertain		1	6-33432168-C-A	1	6.20e-7	-2.827	Likely Benign	0.124	Likely Benign	Likely Benign	0.147	Likely Benign										-0.37	Neutral	0.824	Possibly Damaging	0.880	Possibly Damaging	4.24	Benign	0.00	Affected	4.32	1	3	0	-0.3	-9.01
c.3397A>G	I1133V	Likely Benign		Benign		1	6-33443949-A-G	22	1.48e-5	-3.362	Likely Benign	0.067	Likely Benign	Likely Benign	0.180	Likely Benign										0.06	Neutral	0.007	Benign	0.007	Benign	5.47	Benign	0.58	Tolerated	4.32	3	4	3	-0.3	-14.03																10.1016/j.ajhg.2020.11.011
c.3487C>G	H1163D			Uncertain		1				-2.107	Likely Benign	0.949	Likely Pathogenic	Ambiguous	0.476	Likely Benign										-2.60	Deleterious	0.991	Probably Damaging	0.991	Probably Damaging	5.44	Benign	0.31	Tolerated	3.88	3	1	-1	-0.3	-22.05
c.3502A>G	I1168V	Likely Benign		Uncertain		1				-3.263	Likely Benign	0.524	Ambiguous	Likely Benign	0.363	Likely Benign										-0.14	Neutral	0.876	Possibly Damaging	0.643	Possibly Damaging	5.47	Benign	0.84	Tolerated	3.88	3	4	3	-0.3	-14.03
c.3607C>G	H1203D	Likely Benign	Coiled-coil	Uncertain		1				-6.729	Likely Benign	0.525	Ambiguous	Likely Benign	0.403	Likely Benign										-1.89	Neutral	0.473	Possibly Damaging	0.265	Benign	5.51	Benign	0.24	Tolerated	3.77	5	1	-1	-0.3	-22.05
c.37A>G	I13V	Likely Benign		Uncertain		1				-2.497	Likely Benign	0.105	Likely Benign	Likely Benign	0.110	Likely Benign										0.01	Neutral	0.000	Benign	0.000	Benign	4.25	Benign	0.00	Affected			4	3	-0.3	-14.03
c.467T>G	F156C	Likely Pathogenic		Uncertain		1				-13.658	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.297	Likely Benign										-3.54	Deleterious	0.999	Probably Damaging	0.990	Probably Damaging	3.92	Benign	0.00	Affected			-4	-2	-0.3	-44.04
c.1136C>G	S379W (3D Viewer)		C2	Uncertain		1	6-33438041-C-G			-8.898	Likely Pathogenic	0.388	Ambiguous	Likely Benign	0.520	Likely Pathogenic	4.32	Destabilizing	3.4	3.56	Destabilizing	3.94	Destabilizing	0.16	Likely Benign	-1.02	Neutral	0.998	Probably Damaging	0.844	Possibly Damaging	3.82	Benign	0.01	Affected	4.32	11	-2	-3	-0.1	99.14	271.3	-75.7	1.4	1.0	0.6	0.5								Uncertain	Ser379 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like tryptophan are rarely tolerated. Although no major negative structural effects are observed in the variant simulations, Trp379 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn
c.1154C>G	S385W (3D Viewer)		C2	Benign		1	6-33438059-C-G			-9.353	Likely Pathogenic	0.362	Ambiguous	Likely Benign	0.373	Likely Benign	0.53	Ambiguous	0.2	0.69	Ambiguous	0.61	Ambiguous	0.00	Likely Benign	-0.84	Neutral	0.986	Probably Damaging	0.968	Probably Damaging	4.63	Benign	0.00	Affected	4.32	3	-2	-3	-0.1	99.14	260.4	-71.2	0.5	1.3	0.7	0.4								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like tryptophan are rarely tolerated. Although no major negative structural effects are observed in the variant simulations, Trp385 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effects on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.	10.1016/j.ajhg.2020.11.011
c.1855A>T	T619S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.608	Likely Pathogenic	0.677	Likely Pathogenic	Likely Benign	0.602	Likely Pathogenic	1.09	Ambiguous	0.2	1.35	Ambiguous	1.22	Ambiguous	0.85	Ambiguous	-3.42	Deleterious	0.999	Probably Damaging	0.998	Probably Damaging	-1.30	Pathogenic	0.05	Affected	3.37	35	1	1	-0.1	-14.03
c.1918A>T	T640S (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441177-A-T	1	6.20e-7	-2.371	Likely Benign	0.062	Likely Benign	Likely Benign	0.088	Likely Benign	-0.78	Ambiguous	0.1	0.43	Likely Benign	-0.18	Likely Benign	-0.30	Likely Benign	0.92	Neutral	0.000	Benign	0.001	Benign	3.60	Benign	0.33	Tolerated	3.37	30	1	1	-0.1	-14.03
c.2369C>G	T790S	Likely Benign	SH3-binding motif	Uncertain		1				-3.914	Likely Benign	0.123	Likely Benign	Likely Benign	0.134	Likely Benign										-1.83	Neutral	0.997	Probably Damaging	0.989	Probably Damaging	2.39	Pathogenic	0.33	Tolerated	3.64	6	1	1	-0.1	-14.03
c.2924C>G	T975S	Likely Benign		Uncertain		1				-2.743	Likely Benign	0.068	Likely Benign	Likely Benign	0.109	Likely Benign										-0.57	Neutral	0.059	Benign	0.061	Benign	4.16	Benign	0.20	Tolerated			1	1	-0.1	-14.03
c.667A>T	T223S (3D Viewer)		PH	Conflicting		2	6-33435518-A-T	3	1.86e-6	-7.714	In-Between	0.410	Ambiguous	Likely Benign	0.535	Likely Pathogenic	0.26	Likely Benign	0.1	0.50	Ambiguous	0.38	Likely Benign	0.62	Ambiguous	-2.86	Deleterious	0.421	Benign	0.058	Benign	5.80	Benign	0.02	Affected	3.41	13	1	1	-0.1	-14.03	200.7	17.3	-0.2	0.2	0.0	0.0						X		Uncertain	The introduced residue Ser223 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Its hydroxyl group forms hydrogen bonds with nearby residues Thr228 and Lys207 in the variant simulations, similar to the hydroxyl group of Thr223 in the WT simulations. These hydrogen-bonding interactions at the β sheet surface contribute to the stability of the secondary structure element and may prevent it from unfolding. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.1485A>C	E495D (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-3.574	Likely Benign	0.958	Likely Pathogenic	Likely Pathogenic	0.566	Likely Pathogenic	1.39	Ambiguous	0.1	1.03	Ambiguous	1.21	Ambiguous	0.98	Ambiguous	-2.52	Deleterious	0.998	Probably Damaging	0.989	Probably Damaging	-1.41	Pathogenic	0.17	Tolerated	3.37	35	3	2	0.0	-14.03	220.6	38.8	0.0	0.0	0.1	0.1		X		X				Uncertain	Glu495 is located in the α-helix (res. Leu489-Glu519), and its carboxylate group forms salt bridges with the neighboring Lys492 and with Arg596 on an opposing α-helix (res. Glu582-Met603) in the WT simulations. In the variant simulations, the acidic carboxylate side chain of Asp495 can also form salt bridges with both Lys492 and Arg596. However, the shorter side chain of aspartate tends to favor forming a salt bridge with the nearby Arg499 on the same α-helix instead. Asp495 might not maintain the salt bridge with Arg596 on the opposing α-helix as efficiently as Glu495 in the WT, potentially weakening the tertiary structure. Regardless, the potential negative effect is likely to be minor, with no deleterious effects observed on the protein structure during the simulations. However, due to its location at the GAP-Ras interface, the effect of the residue swap on SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.1851G>T	E617D (3D Viewer)	Likely Benign	GAP	Uncertain		1				-1.349	Likely Benign	0.241	Likely Benign	Likely Benign	0.322	Likely Benign	0.12	Likely Benign	0.1	0.80	Ambiguous	0.46	Likely Benign	0.07	Likely Benign	-0.01	Neutral	0.994	Probably Damaging	0.979	Probably Damaging	-1.35	Pathogenic	0.88	Tolerated	3.37	35	2	3	0.0	-14.03
c.187G>C	E63Q	Likely Benign		Uncertain		1				-4.038	Likely Benign	0.687	Likely Pathogenic	Likely Benign	0.078	Likely Benign										-0.85	Neutral	0.659	Possibly Damaging	0.775	Possibly Damaging	3.90	Benign	0.00	Affected	4.32	1	2	2	0.0	-0.98
c.2050G>A	D684N (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.155	Likely Pathogenic	0.985	Likely Pathogenic	Likely Pathogenic	0.382	Likely Benign	1.47	Ambiguous	0.8	1.76	Ambiguous	1.62	Ambiguous	0.37	Likely Benign	-4.99	Deleterious	0.999	Probably Damaging	0.746	Possibly Damaging	3.39	Benign	0.01	Affected			2	1	0.0	-0.98
c.2158G>A	D720N (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441623-G-A	5	3.10e-6	-9.135	Likely Pathogenic	0.654	Likely Pathogenic	Likely Benign	0.289	Likely Benign	0.01	Likely Benign	0.0	-0.20	Likely Benign	-0.10	Likely Benign	0.46	Likely Benign	-3.74	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	2.18	Pathogenic	0.01	Affected	3.50	9	1	2	0.0	-0.98
c.2215G>C	E739Q	Likely Benign		Uncertain		1				-2.846	Likely Benign	0.161	Likely Benign	Likely Benign	0.071	Likely Benign										-1.06	Neutral	0.801	Possibly Damaging	0.339	Benign	2.57	Benign	0.00	Affected	4.32	2	2	2	0.0	-0.98
c.2217G>C	E739D	Likely Benign		Uncertain		1				-3.369	Likely Benign	0.062	Likely Benign	Likely Benign	0.097	Likely Benign										-0.49	Neutral	0.002	Benign	0.005	Benign	2.59	Benign	0.00	Affected			3	2	0.0	-14.03
c.2302G>A	D768N	Likely Benign		Uncertain		1	6-33442460-G-A	2	2.57e-6	-6.892	Likely Benign	0.453	Ambiguous	Likely Benign	0.048	Likely Benign										-0.77	Neutral	0.106	Benign	0.009	Benign	4.07	Benign	0.96	Tolerated	3.64	6	1	2	0.0	-0.98
c.1966G>C	E656Q (3D Viewer)		GAP	Uncertain		1	6-33441225-G-C	1	6.20e-7	-9.145	Likely Pathogenic	0.766	Likely Pathogenic	Likely Benign	0.249	Likely Benign	-0.14	Likely Benign	0.0	-0.81	Ambiguous	-0.48	Likely Benign	0.25	Likely Benign	-2.29	Neutral	0.980	Probably Damaging	0.528	Possibly Damaging	3.46	Benign	0.02	Affected	3.39	24	2	2	0.0	-0.98	224.3	1.7	0.0	0.1	0.1	0.0		X						Potentially Benign	The carboxylate side chain of Glu656, located on an α helix (res. Ser641-Glu666), frequently forms a hydrogen bond with the nearby residue Ser659 on the same α helix. In the variant simulations, the carboxamide side chain of Gln656 alternatively forms a hydrogen bond with either Ser659 or Glu548 on an opposing helix (res. Ala533-Val560).Although the frequent interaction between Gln656 and Glu548 may strengthen or stabilize the tertiary structure assembly, the effect is likely to be marginal.
c.1998G>C	E666D (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.820	Likely Pathogenic	0.704	Likely Pathogenic	Likely Benign	0.197	Likely Benign	0.88	Ambiguous	0.0	0.37	Likely Benign	0.63	Ambiguous	1.05	Destabilizing	-2.69	Deleterious	0.992	Probably Damaging	0.603	Possibly Damaging	3.43	Benign	0.06	Tolerated	3.38	28	3	2	0.0	-14.03	237.2	16.5	0.0	0.0	-0.3	0.1		X						Potentially Pathogenic	The carboxylate group of Glu666, located on the α-helix (res. Ser641-Glu666), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), such as Lys566, Thr672, and Asn669, in the WT simulations. In the variant simulations, the shorter side chain of Asp666 cannot maintain these interactions as efficiently as Glu666 in the WT, resulting in a less coordinated hydrogen-bond network.
c.2493G>C	E831D	Likely Benign		Uncertain		1	6-33443045-G-C	1	6.19e-7	-3.055	Likely Benign	0.063	Likely Benign	Likely Benign	0.073	Likely Benign										1.23	Neutral	0.002	Benign	0.002	Benign	2.64	Benign	0.77	Tolerated	3.77	5	3	2	0.0	-14.03
c.291G>T	E97D	Likely Benign		Uncertain		3	6-33425899-G-T			-3.239	Likely Benign	0.077	Likely Benign	Likely Benign	0.081	Likely Benign										-0.49	Neutral	0.880	Possibly Damaging	0.636	Possibly Damaging	4.12	Benign	0.00	Affected	4.32	1	3	2	0.0	-14.03
c.3022G>A	D1008N	Likely Benign		Likely Benign		1	6-33443574-G-A	3	1.86e-6	-4.045	Likely Benign	0.714	Likely Pathogenic	Likely Benign	0.128	Likely Benign										-2.15	Neutral	0.999	Probably Damaging	0.997	Probably Damaging	2.75	Benign	0.01	Affected	3.77	5	2	1	0.0	-0.98
c.3048C>A	D1016E	Likely Benign		Likely Benign		1	6-33443600-C-A	2	1.24e-6	-3.422	Likely Benign	0.216	Likely Benign	Likely Benign	0.017	Likely Benign										-0.37	Neutral	0.008	Benign	0.028	Benign	2.64	Benign	0.65	Tolerated	3.77	5	2	3	0.0	14.03
c.3567G>C	E1189D	Likely Benign	Coiled-coil	Likely Benign		1	6-33444602-G-C	3	1.86e-6	-3.582	Likely Benign	0.461	Ambiguous	Likely Benign	0.359	Likely Benign										-1.42	Neutral	0.992	Probably Damaging	0.989	Probably Damaging	5.30	Benign	0.25	Tolerated	3.82	4	3	2	0.0	-14.03
c.3846G>C	E1282D	Likely Benign		Uncertain		1	6-33447894-G-C	1	6.44e-7	-3.879	Likely Benign	0.074	Likely Benign	Likely Benign	0.104	Likely Benign										-1.26	Neutral	0.112	Benign	0.036	Benign	2.70	Benign	0.39	Tolerated	3.77	5	3	2	0.0	-14.03
c.3858A>T	E1286D	Likely Benign		Conflicting		4	6-33447906-A-T	143	9.22e-5	-4.010	Likely Benign	0.081	Likely Benign	Likely Benign	0.036	Likely Benign										1.02	Neutral	0.001	Benign	0.004	Benign	2.96	Benign	1.00	Tolerated	3.77	5	3	2	0.0	-14.03																10.1016/j.ajhg.2020.11.011
c.4000A>G	N1334D			Uncertain		1	6-33451874-A-G			-4.584	Likely Benign	0.674	Likely Pathogenic	Likely Benign	0.126	Likely Benign										-3.06	Deleterious	0.886	Possibly Damaging	0.522	Possibly Damaging	3.55	Benign	0.00	Affected	3.77	5	1	2	0.0	0.98
c.4008G>C	E1336D	Likely Benign		Likely Benign		1				-3.344	Likely Benign	0.596	Likely Pathogenic	Likely Benign	0.062	Likely Benign										-1.92	Neutral	0.001	Benign	0.003	Benign	3.30	Benign	0.00	Affected	3.77	5	2	3	0.0	-14.03
c.453C>A	D151E	Likely Benign		Uncertain		1				-5.662	Likely Benign	0.886	Likely Pathogenic	Ambiguous	0.142	Likely Benign										-2.02	Neutral	0.984	Probably Damaging	0.967	Probably Damaging	3.99	Benign	0.11	Tolerated	3.61	5	3	2	0.0	14.03
c.505G>A	D169N			Uncertain		1				-10.713	Likely Pathogenic	0.761	Likely Pathogenic	Likely Benign	0.110	Likely Benign										-2.04	Neutral	0.079	Benign	0.052	Benign	4.07	Benign	0.01	Affected	3.74	4	2	1	0.0	-0.98
c.603T>A	D201E (3D Viewer)	Likely Benign	PH	Benign		1				-2.640	Likely Benign	0.406	Ambiguous	Likely Benign	0.165	Likely Benign	0.42	Likely Benign	0.2	1.99	Ambiguous	1.21	Ambiguous	0.23	Likely Benign	-0.69	Neutral	0.633	Possibly Damaging	0.108	Benign	4.30	Benign	1.00	Tolerated	3.46	9	3	2	0.0	14.03	258.7	-24.8	0.9	0.1	-0.3	0.2						X		Uncertain	Asp201, an acidic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another acidic residue, glutamate. The carboxylate groups of both Asp201 and Glu201 side chains form hydrogen bonds with the hydroxyl group of Ser221 in the simulations. Due to its shorter side chain, Asp201 can also hydrogen bond with the backbone amide groups of neighboring loop residues Ser204 and Asp203. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.603T>G	D201E (3D Viewer)	Likely Benign	PH	Conflicting		2	6-33435245-T-G	20	1.24e-5	-2.640	Likely Benign	0.406	Ambiguous	Likely Benign	0.165	Likely Benign	0.42	Likely Benign	0.2	1.99	Ambiguous	1.21	Ambiguous	0.23	Likely Benign	-0.69	Neutral	0.633	Possibly Damaging	0.108	Benign	4.30	Benign	1.00	Tolerated	3.46	9	3	2	0.0	14.03	258.7	-24.8	0.9	0.1	-0.3	0.2						X		Uncertain	Asp201, an acidic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another acidic residue, glutamate. The carboxylate groups of both Asp201 and Glu201 side chains form hydrogen bonds with the hydroxyl group of Ser221 in the simulations. Due to its shorter side chain, Asp201 can also hydrogen bond with the backbone amide groups of neighboring loop residues Ser204 and Asp203. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.718G>A	D240N	Likely Pathogenic	PH	Uncertain		1				-12.942	Likely Pathogenic	0.755	Likely Pathogenic	Likely Benign	0.701	Likely Pathogenic	0.22	Likely Benign	0.9	0.47	Likely Benign	0.35	Likely Benign	0.37	Likely Benign	-4.37	Deleterious	0.993	Probably Damaging	0.984	Probably Damaging	5.88	Benign	0.01	Affected			2	1	0.0	-0.98
c.862G>A	D288N (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437767-G-A	2	1.24e-6	-10.535	Likely Pathogenic	0.521	Ambiguous	Likely Benign	0.321	Likely Benign	-0.39	Likely Benign	0.1	0.01	Likely Benign	-0.19	Likely Benign	-0.03	Likely Benign	-3.73	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.78	Pathogenic	0.05	Affected	3.38	23	1	2	0.0	-0.98
c.910G>A	D304N (3D Viewer)		C2	Uncertain		1				-6.194	Likely Benign	0.391	Ambiguous	Likely Benign	0.345	Likely Benign	0.30	Likely Benign	0.1	-0.08	Likely Benign	0.11	Likely Benign	0.21	Likely Benign	-4.18	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.81	Pathogenic	0.03	Affected	3.38	23	1	2	0.0	-0.98
c.819G>T	E273D (3D Viewer)	Likely Benign	C2	Benign		1	6-33437724-G-T	2	1.24e-6	-1.811	Likely Benign	0.058	Likely Benign	Likely Benign	0.092	Likely Benign	0.26	Likely Benign	0.1	-0.48	Likely Benign	-0.11	Likely Benign	-0.63	Ambiguous	1.99	Neutral	0.004	Benign	0.010	Benign	2.00	Pathogenic	1.00	Tolerated	3.38	18	3	2	0.0	-14.03	223.1	22.1	0.2	0.0	0.0	0.1						X		Potentially Benign	The negatively charged residue Glu273, located in a β hairpin loop (res. Glu273-Lys278) that connects two anti-parallel β sheet strands, is replaced with another negatively charged residue, aspartate. Because the C2 domain loop faces the membrane surface, the potentially crucial role of the carboxylate group of Glu273 or Asp273 on SynGAP-membrane association cannot be fully explored via solvent-only simulations.As a minor note, the neighboring residue Arg272, which stacks with the indole ring of the Trp362 side chain and directly faces RasGTPase, forms a salt bridge more often with Asp273 than with the non-mutated Glu273 in the simulations. Regardless, due to the similar physicochemical properties of the WT and variant residues at the membrane interface, the residue swap is likely to be well tolerated.
c.930G>C	E310D (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-11.218	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.666	Likely Pathogenic	1.87	Ambiguous	0.5	2.39	Destabilizing	2.13	Destabilizing	1.04	Destabilizing	-2.76	Deleterious	0.997	Probably Damaging	0.992	Probably Damaging	1.19	Pathogenic	0.02	Affected	3.38	19	3	2	0.0	-14.03	232.6	27.2	0.1	0.0	0.1	0.1						X		Potentially Benign	The carboxylate group of Glu310, located in an anti-parallel β sheet strand (res. Thr305-Asn315), is ideally positioned to interact with the hydroxyl and backbone amide groups of Thr295 on a twisted anti-parallel β strand. Because the carboxylate group can also interact with the GAP domain residues (e.g., Gln612, Tyr614), Glu310 potentially plays a key role in maintaining the tertiary assembly between the C2 and GAP domains. In the variant simulations, the carboxylate group of Asp310 can form the same interactions as glutamate; however, due to its one hydrocarbon shorter length, the connections are less stable or less optimal.
c.1604G>C	S535T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33438847-G-C	14	8.67e-6	-3.886	Likely Benign	0.069	Likely Benign	Likely Benign	0.177	Likely Benign	0.45	Likely Benign	0.1	-0.27	Likely Benign	0.09	Likely Benign	0.17	Likely Benign	-0.81	Neutral	0.000	Benign	0.001	Benign	-1.25	Pathogenic	0.25	Tolerated	3.37	35	1	1	0.1	14.03	201.3	-17.3	-0.1	0.7	-0.2	0.1						X		Potentially Benign	Ser535 is located near the terminal end of an α-helix (res. Ala533-Val560) close to the membrane interface. In the WT simulations, the hydroxyl side chain of Ser535 forms hydrogen bonds with nearby residues (e.g., His539, Glu538) without any specific interactions. These hydrogen bonds disrupt the structure of the terminal end of the α-helix (Ala533-Ser535), causing it to weaken or unfold during the WT simulations. In the variant simulations, Thr535, a hydrophilic residue with a hydroxyl group of almost the same size as Ser, interacts more frequently with the preceding loop residues (e.g., Thr532, Cys531) due to its longer side chain. Regardless, the residue swap is tolerated in the simulations with no negative effects. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.	10.1016/j.ajhg.2020.11.011
c.2111G>C	S704T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.930	Likely Benign	0.265	Likely Benign	Likely Benign	0.071	Likely Benign	0.80	Ambiguous	0.0	0.15	Likely Benign	0.48	Likely Benign	0.29	Likely Benign	-1.72	Neutral	0.525	Possibly Damaging	0.107	Benign	3.45	Benign	0.07	Tolerated	3.47	10	1	1	0.1	14.03	201.7	-18.0	0.0	0.0	-0.2	0.7						X		Potentially Benign	Ser704 is located at the end and outer surface of an α-helix (res. Thr704-Gly712), which is connected via a tight turn or loop to another α-helix (res. Asp684-Gln702). The hydroxyl side chain of Ser704 occasionally forms a hydrogen bond with the amide group of Ala707. Similarly, in the variant simulations, the hydroxyl side chain of Thr704 forms hydrogen bonds with the amide groups of Ala707 and Leu708. Thus, the residue swap does not cause any apparent structural change.
c.1715G>C	W572S (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-17.461	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.775	Likely Pathogenic	5.78	Destabilizing	0.2	3.37	Destabilizing	4.58	Destabilizing	1.79	Destabilizing	-12.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.24	Pathogenic	0.01	Affected	3.37	35	-2	-3	0.1	-99.14	235.1	76.6	0.0	0.0	-0.4	0.1							X	Potentially Pathogenic	The introduced residue Ser572, located in an α-helix (res. Arg563-Glu578), is considerably smaller than the tryptophan it replaced. The indole ring of the Trp572 side chain lies in a hydrophobic inter-helix space, where it makes extensive hydrophobic interactions with nearby residues such as Met470, Phe569, Leu588, and Ile589. In the variant simulations, all these favorable packing interactions are completely removed, as the introduced residue Ser572 is too hydrophilic or small to fill the hydrophobic niche occupied by the indole ring. Moreover, the hydroxyl group of Ser572 forms hydrogen bonds with the carbonyl groups of Glu567 and Val568 within the same α-helix, potentially lowering its integrity. Overall, the residue swap is highly likely to cause critical protein folding problems that are underestimated based on the effects seen in the variant simulations.
c.2362T>A	S788T	Likely Benign	SH3-binding motif	Uncertain		2	6-33442914-T-A	4	2.49e-6	-4.288	Likely Benign	0.288	Likely Benign	Likely Benign	0.092	Likely Benign										-2.25	Neutral	0.979	Probably Damaging	0.982	Probably Damaging	1.55	Pathogenic	0.02	Affected	3.64	6	1	1	0.1	14.03
c.1027G>A	V343I (3D Viewer)	Likely Benign	C2	Uncertain		2	6-33437932-G-A	1	6.20e-7	-6.020	Likely Benign	0.117	Likely Benign	Likely Benign	0.020	Likely Benign	-0.27	Likely Benign	0.0	-0.04	Likely Benign	-0.16	Likely Benign	-0.39	Likely Benign	-0.14	Neutral	0.159	Benign	0.084	Benign	1.98	Pathogenic	0.27	Tolerated	3.37	25	4	3	0.3	14.03	240.2	-26.9	-0.2	0.2	-0.2	0.2						X		Potentially Benign	The iso-propyl side chain of Val343, located in an anti-parallel β sheet strand (res. Gly341-Pro349), is packing against multiple hydrophobic residues of the C2 domain (e.g., Leu327, Leu274, Val365). In the variant simulations, the sec-butyl side chain of Ile343 is basically able to form the same interactions as valine due to its similar hydrophobic profile. The residue swap also does not seem to cause negative effects on the protein structure based on the simulations.
c.103G>A	V35I	Likely Benign		Uncertain		1	6-33423512-G-A	5	3.10e-6	-3.764	Likely Benign	0.081	Likely Benign	Likely Benign	0.017	Likely Benign										-0.32	Neutral	0.672	Possibly Damaging	0.369	Benign	4.16	Benign	0.00	Affected	4.32	1	3	4	0.3	14.03
c.1221G>T	Q407H (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-10.526	Likely Pathogenic	0.830	Likely Pathogenic	Ambiguous	0.206	Likely Benign	0.59	Ambiguous	0.0	0.61	Ambiguous	0.60	Ambiguous	1.10	Destabilizing	-4.51	Deleterious	0.982	Probably Damaging	0.947	Probably Damaging	3.88	Benign	0.01	Affected	3.38	28	0	3	0.3	9.01
c.1300G>A	V434I (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438205-G-A	1	6.19e-7	-6.999	Likely Benign	0.129	Likely Benign	Likely Benign	0.192	Likely Benign	-0.04	Likely Benign	0.0	0.22	Likely Benign	0.09	Likely Benign	0.31	Likely Benign	-0.82	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.53	Benign	0.18	Tolerated	3.37	29	4	3	0.3	14.03	246.7	-27.7	0.0	0.0	0.1	0.0						X		Potentially Benign	The iso-propyl side chain of Val434, located at the end of an α helix (res. Met414-Glu436), packs against hydrophobic residues in an interhelix space (e.g., Met430, Ala707, Leu711). In the variant simulations, the sec-butyl group of Ile434 is able to form the same hydrophobic interactions. Accordingly, the residue swap does not negatively affect the protein structure based on the simulations.
c.1354G>A	V452I (3D Viewer)		GAP	Uncertain		1				-8.985	Likely Pathogenic	0.361	Ambiguous	Likely Benign	0.218	Likely Benign	-0.08	Likely Benign	0.1	0.51	Ambiguous	0.22	Likely Benign	0.25	Likely Benign	-0.99	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.26	Benign	0.05	Affected			4	3	0.3	14.03
c.1417G>A	V473I (3D Viewer)		GAP	Uncertain		1	6-33438449-G-A	1	6.20e-7	-7.481	In-Between	0.418	Ambiguous	Likely Benign	0.203	Likely Benign	-0.12	Likely Benign	0.0	1.20	Ambiguous	0.54	Ambiguous	-0.06	Likely Benign	-0.91	Neutral	0.929	Possibly Damaging	0.917	Probably Damaging	3.74	Benign	0.18	Tolerated	3.37	34	3	4	0.3	14.03
c.1663G>A	V555I (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.544	Likely Benign	0.084	Likely Benign	Likely Benign	0.253	Likely Benign	-0.82	Ambiguous	0.0	-0.41	Likely Benign	-0.62	Ambiguous	-0.55	Ambiguous	0.45	Neutral	0.002	Benign	0.002	Benign	-1.26	Pathogenic	1.00	Tolerated			4	3	0.3	14.03
c.2050G>C	D684H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.194	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.613	Likely Pathogenic	3.36	Destabilizing	1.0	2.95	Destabilizing	3.16	Destabilizing	0.55	Ambiguous	-6.98	Deleterious	1.000	Probably Damaging	0.972	Probably Damaging	3.36	Benign	0.00	Affected	3.42	17	-1	1	0.3	22.05
c.256G>A	V86I	Likely Benign		Uncertain		1				-4.726	Likely Benign	0.338	Likely Benign	Likely Benign	0.076	Likely Benign										-0.31	Neutral	0.267	Benign	0.097	Benign	3.94	Benign	0.00	Affected	4.32	1	4	3	0.3	14.03
c.2578G>A	V860I	Likely Benign		Benign		1	6-33443130-G-A	21	1.30e-5	-4.516	Likely Benign	0.095	Likely Benign	Likely Benign	0.039	Likely Benign										-0.42	Neutral	0.009	Benign	0.006	Benign	4.24	Benign	0.00	Affected	3.77	5	4	3	0.3	14.03
c.2596G>A	V866I	Likely Benign		Conflicting		3	6-33443148-G-A	5	3.10e-6	-4.652	Likely Benign	0.118	Likely Benign	Likely Benign	0.059	Likely Benign										-0.39	Neutral	0.957	Probably Damaging	0.541	Possibly Damaging	2.69	Benign	0.27	Tolerated	3.82	4	4	3	0.3	14.03
c.2724G>C	Q908H	Likely Benign		Conflicting		4	6-33443276-G-C	1	6.20e-7	-4.658	Likely Benign	0.311	Likely Benign	Likely Benign	0.112	Likely Benign										-0.74	Neutral	0.996	Probably Damaging	0.995	Probably Damaging	2.58	Benign	0.05	Affected	3.77	5	3	0	0.3	9.01
c.2809G>C	D937H	Likely Benign		Uncertain		1				-0.733	Likely Benign	0.677	Likely Pathogenic	Likely Benign	0.150	Likely Benign										-1.74	Neutral	1.000	Probably Damaging	0.975	Probably Damaging	2.68	Benign	0.13	Tolerated	3.77	5	-1	1	0.3	22.05
c.3192G>C	Q1064H	Likely Benign		Uncertain		1				-4.576	Likely Benign	0.162	Likely Benign	Likely Benign	0.063	Likely Benign										-0.66	Neutral	0.938	Possibly Damaging	0.596	Possibly Damaging	4.15	Benign	0.05	Affected			3	0	0.3	9.01
c.451G>C	D151H	Likely Pathogenic		Uncertain		1	6-33432748-G-C	2	1.26e-6	-11.747	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.335	Likely Benign										-3.90	Deleterious	0.999	Probably Damaging	0.995	Probably Damaging	3.86	Benign	0.00	Affected	3.61	5	-1	1	0.3	22.05
c.70G>A	V24I	Likely Benign		Uncertain		1	6-33423479-G-A	9	5.58e-6	-3.701	Likely Benign	0.137	Likely Benign	Likely Benign	0.069	Likely Benign										-0.25	Neutral	0.043	Benign	0.031	Benign	3.96	Benign	0.00	Affected	4.32	1	3	4	0.3	14.03
c.958G>A	V320I (3D Viewer)	Likely Benign	C2	Uncertain		1				-5.220	Likely Benign	0.111	Likely Benign	Likely Benign	0.027	Likely Benign	-0.27	Likely Benign	0.2	0.66	Ambiguous	0.20	Likely Benign	0.01	Likely Benign	-0.21	Neutral	0.198	Benign	0.114	Benign	1.77	Pathogenic	0.45	Tolerated	3.38	23	3	4	0.3	14.03
c.859G>C	D287H (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-14.518	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.589	Likely Pathogenic	0.48	Likely Benign	0.3	0.32	Likely Benign	0.40	Likely Benign	0.63	Ambiguous	-6.43	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.51	Pathogenic	0.00	Affected	3.38	23	1	-1	0.3	22.05	235.6	3.8	0.1	1.2	0.1	0.1	X	X						Potentially Pathogenic	The carboxylate group of Asp287, located at the beginning of a β hairpin loop connecting two anti-parallel β sheet strands (res. Arg279-Leu286, res. Met289-Pro298), maintains a salt bridge with the guanidinium group of Arg324 in the β sheet during the WT simulations. In the variant simulations, the imidazole ring of the His287 side chain is unable to form a salt bridge with Arg324 or establish any other stable compensatory interactions, which could weaken the beta sandwich assembly of the C2 domain. This destabilization of the C2 domain could adversely affect the stability of the SynGAP-membrane association.
c.1345A>G	S449G (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438250-A-G	3	1.86e-6	-5.936	Likely Benign	0.071	Likely Benign	Likely Benign	0.116	Likely Benign	0.47	Likely Benign	0.0	0.55	Ambiguous	0.51	Ambiguous	0.85	Ambiguous	-2.32	Neutral	0.948	Possibly Damaging	0.124	Benign	3.35	Benign	0.13	Tolerated	3.37	32	0	1	0.4	-30.03
c.1474A>G	K492E (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-16.175	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.510	Likely Pathogenic	1.53	Ambiguous	0.1	1.90	Ambiguous	1.72	Ambiguous	1.42	Destabilizing	-3.98	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.99	Benign	0.01	Affected	3.37	35	1	0	0.4	0.94
c.1606T>G	L536V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.014	Likely Pathogenic	0.269	Likely Benign	Likely Benign	0.586	Likely Pathogenic	1.25	Ambiguous	0.3	1.22	Ambiguous	1.24	Ambiguous	1.20	Destabilizing	-2.81	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	-1.34	Pathogenic	0.09	Tolerated	3.37	34	2	1	0.4	-14.03	204.7	26.4	0.2	0.0	-0.2	0.2						X		Potentially Benign	Leu536 is located on an α-helix (res. Ala533-Val560) at the membrane interface. The iso-butyl group of Leu536 interacts with nearby hydrophobic residues in the preceding loop (e.g., Val526, Pro528, Cys531). In the variant simulations, the iso-propyl side chain of Val536 forms similar hydrophobic interactions as Leu536 in the WT, causing no negative structural effects.
c.1393C>G	L465V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.893	Likely Pathogenic	0.838	Likely Pathogenic	Ambiguous	0.276	Likely Benign	2.46	Destabilizing	0.1	2.66	Destabilizing	2.56	Destabilizing	1.21	Destabilizing	-2.98	Deleterious	0.996	Probably Damaging	0.992	Probably Damaging	2.44	Pathogenic	0.10	Tolerated	3.37	34	2	1	0.4	-14.03	204.3	30.9	0.0	0.0	-0.4	0.6						X		Potentially Benign	The iso-butyl side chain of Leu465, located in the middle of an α helix (res. Ala461–Phe476), packs with hydrophobic residues (e.g., Phe464, Met468, Tyr497, Ile494) in an inter-helix space formed with two other α helices (res. Ala461–Phe476 and res. Thr488-Gly502). In the variant simulations, the iso-propyl side chain of Val465 is equally sized and similarly hydrophobic as the original side chain of Leu465. Hence, the mutation does not exert any negative effects on the protein structure based on the variant simulations.
c.1792C>G	L598V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.002	Likely Pathogenic	0.578	Likely Pathogenic	Likely Benign	0.221	Likely Benign	1.89	Ambiguous	0.1	1.58	Ambiguous	1.74	Ambiguous	1.01	Destabilizing	-2.92	Deleterious	0.944	Possibly Damaging	0.786	Possibly Damaging	3.21	Benign	0.02	Affected	3.37	35	2	1	0.4	-14.03	218.4	29.6	0.0	0.0	0.8	0.0						X		Potentially Benign	The iso-butyl side chain of Leu598, located on an α helix (res. Glu582-Met603), packs hydrophobically with other hydrophobic residues in the inter-helix space (e.g., Ile602, Phe594, Ile510).In the variant simulations, Val598, which has similar size and physicochemical properties to leucine, resides in the inter-helix hydrophobic space in a similar manner to Leu598 in the WT. This causes no negative effects on the protein structure.
c.1873C>G	L625V	Likely Pathogenic	GAP	Uncertain		1				-11.319	Likely Pathogenic	0.833	Likely Pathogenic	Ambiguous	0.480	Likely Benign	1.80	Ambiguous	0.7	1.69	Ambiguous	1.75	Ambiguous	1.42	Destabilizing	-2.96	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	3.07	Benign	0.01	Affected			2	1	0.4	-14.03
c.1957C>G	L653V	Likely Benign	GAP	Uncertain		1				-7.050	In-Between	0.301	Likely Benign	Likely Benign	0.146	Likely Benign	3.28	Destabilizing	0.3	2.18	Destabilizing	2.73	Destabilizing	1.32	Destabilizing	-2.25	Neutral	0.227	Benign	0.039	Benign	3.28	Benign	0.08	Tolerated			2	1	0.4	-14.03
c.2086C>G	L696V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.909	Likely Pathogenic	0.745	Likely Pathogenic	Likely Benign	0.351	Likely Benign	2.35	Destabilizing	0.1	1.85	Ambiguous	2.10	Destabilizing	1.46	Destabilizing	-2.79	Deleterious	0.992	Probably Damaging	0.970	Probably Damaging	3.16	Benign	0.00	Affected	3.46	13	1	2	0.4	-14.03
c.2113A>C	K705Q (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441372-A-C	1	6.20e-7	-5.787	Likely Benign	0.436	Ambiguous	Likely Benign	0.142	Likely Benign	-0.10	Likely Benign	0.0	0.33	Likely Benign	0.12	Likely Benign	-0.02	Likely Benign	-0.24	Neutral	0.997	Probably Damaging	0.969	Probably Damaging	3.42	Benign	0.78	Tolerated	3.47	10	1	1	0.4	-0.04
c.2115G>C	K705N (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-9.767	Likely Pathogenic	0.925	Likely Pathogenic	Ambiguous	0.183	Likely Benign	0.74	Ambiguous	0.0	0.37	Likely Benign	0.56	Ambiguous	0.44	Likely Benign	-3.12	Deleterious	0.996	Probably Damaging	0.876	Possibly Damaging	3.37	Benign	0.02	Affected	3.47	10	1	0	0.4	-14.07	221.4	-20.2	0.0	0.0	0.0	0.1						X		Uncertain	The amino side chain of Lys705, located at the end and outer surface of an α-helix (res. Thr704-Gly712), does not form any interactions in the WT simulations. In the variant simulations, the carboxamide side chain of Asn705 briefly forms a salt bridge with Glu706. However, there is no apparent difference between the systems. Due to the model ending abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2131C>G	L711V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33441596-C-G	1	6.20e-7	-10.045	Likely Pathogenic	0.709	Likely Pathogenic	Likely Benign	0.170	Likely Benign	3.48	Destabilizing	0.1	2.22	Destabilizing	2.85	Destabilizing	1.40	Destabilizing	-2.59	Deleterious	0.992	Probably Damaging	0.970	Probably Damaging	3.34	Benign	0.00	Affected	3.50	9	1	2	0.4	-14.03
c.1768A>G	S590G (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2	6-33440820-A-G	14	8.67e-6	-14.277	Likely Pathogenic	0.574	Likely Pathogenic	Likely Benign	0.379	Likely Benign	0.67	Ambiguous	0.1	1.28	Ambiguous	0.98	Ambiguous	0.71	Ambiguous	-3.92	Deleterious	1.000	Probably Damaging	0.922	Probably Damaging	3.42	Benign	0.06	Tolerated	3.37	35	1	0	0.4	-30.03	186.7	49.4	0.0	0.0	0.1	0.0		X						Potentially Pathogenic	In the WT simulations, the hydroxyl group of Ser590, located on an α helix (res. Glu582-Met603), forms hydrogen bonds with the backbone carbonyl of Ala634 and/or the carboxamide group of the Asn635 side chain at the end of the opposing α helix (res. Thr619-Ala634).The residue swap could weaken the integrity of the α helix, as glycine is known as an “α helix breaker.” However, no discernible difference was observed between the WT and variant simulations in this regard. Importantly, Gly590 cannot form hydrogen bonds with the opposing helix in the same way that serine can, which could weaken the tertiary structure assembly between the two helices.
c.1819C>G	L607V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33440871-C-G	2	1.24e-6	-11.190	Likely Pathogenic	0.637	Likely Pathogenic	Likely Benign	0.715	Likely Pathogenic	1.04	Ambiguous	0.2	1.36	Ambiguous	1.20	Ambiguous	0.90	Ambiguous	-2.99	Deleterious	0.985	Probably Damaging	0.992	Probably Damaging	-1.50	Pathogenic	0.01	Affected	3.37	35	2	1	0.4	-14.03	216.3	28.1	0.1	0.0	0.9	0.2				X				Potentially Benign	Leu607 is located in a short helical region (res. Ser606-Phe608) within an α-α loop connecting two α helices (res. Glu582-Met603 and res. Glu617-Asn635). In the WT simulations, the iso-butyl side chain of Leu607 does not interact with any other residues, but it could potentially interact directly with Ras due to its location at the GAP domain.In the variant simulations, Val607, which has similar size and physicochemical properties to leucine, does not cause any negative effects on the protein structure. However, due to its location at the GAP-Ras interface, the residue swap could affect the complex formation with the GTPase, but this cannot be investigated using solvent-only simulations.
c.2506A>G	S836G	Likely Benign		Uncertain		1	6-33443058-A-G	4	2.48e-6	-4.749	Likely Benign	0.112	Likely Benign	Likely Benign	0.066	Likely Benign										-1.65	Neutral	0.006	Benign	0.019	Benign	2.54	Benign	0.39	Tolerated	3.77	5	1	0	0.4	-30.03
c.2608C>G	L870V	Likely Benign		Uncertain		1				-4.123	Likely Benign	0.300	Likely Benign	Likely Benign	0.111	Likely Benign										-1.19	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.64	Benign	0.12	Tolerated	3.88	3	2	1	0.4	-14.03
c.304T>G	L102V	Likely Benign		Uncertain		1	6-33432169-T-G	1	6.20e-7	-4.316	Likely Benign	0.068	Likely Benign	Likely Benign	0.102	Likely Benign										0.32	Neutral	0.880	Possibly Damaging	0.899	Possibly Damaging	4.21	Benign	0.00	Affected	4.32	1	2	1	0.4	-14.03
c.3262A>G	S1088G	Likely Benign		Uncertain		1				-5.034	Likely Benign	0.285	Likely Benign	Likely Benign	0.163	Likely Benign										-1.83	Neutral	0.979	Probably Damaging	0.973	Probably Damaging	2.63	Benign	0.03	Affected	3.77	5	0	1	0.4	-30.03
c.3328A>G	S1110G	Likely Benign		Likely Benign		1				-4.674	Likely Benign	0.079	Likely Benign	Likely Benign	0.035	Likely Benign										-2.26	Neutral	0.036	Benign	0.026	Benign	2.19	Pathogenic	0.08	Tolerated	4.32	2	1	0	0.4	-30.03
c.3361A>G	S1121G	Likely Benign		Uncertain		1	6-33443913-A-G	1	7.00e-7	-1.220	Likely Benign	0.054	Likely Benign	Likely Benign	0.067	Likely Benign										-0.53	Neutral	0.003	Benign	0.004	Benign	6.63	Benign	0.00	Affected	3.77	5	0	1	0.4	-30.03
c.3405G>C	K1135N	Likely Benign		Uncertain		1				-5.715	Likely Benign	0.960	Likely Pathogenic	Likely Pathogenic	0.166	Likely Benign										-0.97	Neutral	0.411	Benign	0.321	Benign	5.43	Benign	0.07	Tolerated	4.32	2	1	0	0.4	-14.07
c.3508A>G	S1170G	Likely Benign	Coiled-coil	Uncertain		1				-4.288	Likely Benign	0.221	Likely Benign	Likely Benign	0.349	Likely Benign										-0.81	Neutral	0.241	Benign	0.229	Benign	5.31	Benign	0.54	Tolerated	4.32	4	1	0	0.4	-30.03
c.3574C>G	L1192V	Likely Benign	Coiled-coil	Uncertain		1				-4.132	Likely Benign	0.471	Ambiguous	Likely Benign	0.041	Likely Benign										-0.89	Neutral	0.779	Possibly Damaging	0.527	Possibly Damaging	2.69	Benign	0.16	Tolerated			2	1	0.4	-14.03
c.3862A>G	K1288E			Uncertain		1	6-33447910-A-G	5	3.22e-6	-2.751	Likely Benign	0.407	Ambiguous	Likely Benign	0.185	Likely Benign										-3.27	Deleterious	0.979	Probably Damaging	0.973	Probably Damaging	2.13	Pathogenic	0.00	Affected	3.77	5	1	0	0.4	0.94
c.526A>G	S176G			Uncertain		1	6-33435168-A-G	1	6.20e-7	-7.541	In-Between	0.360	Ambiguous	Likely Benign	0.066	Likely Benign										-1.08	Neutral	0.131	Benign	0.039	Benign	4.08	Benign	0.22	Tolerated	3.54	6	0	1	0.4	-30.03
c.762G>C	K254N (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-13.306	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.757	Likely Pathogenic	0.73	Ambiguous	0.2	1.87	Ambiguous	1.30	Ambiguous	1.19	Destabilizing	-4.23	Deleterious	0.384	Benign	0.070	Benign	5.93	Benign	0.01	Affected	3.39	15	1	0	0.4	-14.07	215.3	-21.0	-1.0	1.7	0.2	0.3		X						Potentially Pathogenic	The amino group of Lys254, located in an α-β loop connecting the PH and C2 domains (res. Lys251-Arg258), forms salt bridges with the carboxylate groups of Glu244 and Asp684. Since the neutral carboxamide group of the Asn254 side chain cannot form salt bridges with acidic residues, the residue swap potentially weakens the tertiary structure assembly and/or influences the loop positioning. Regardless, in both the variant and WT simulations, all hydrogen bonds formed by the residue’s side chain were broken, and the residue rotated outwards. The partially α helical conformation of the loop, which extends to a nearby α helix (res. Met414-Asn426), is dynamic, making it unclear if the mutation affects it.
c.1025A>C	Y342S (3D Viewer)	Likely Pathogenic	C2	Uncertain		2				-7.996	In-Between	0.925	Likely Pathogenic	Ambiguous	0.407	Likely Benign	3.03	Destabilizing	0.1	2.87	Destabilizing	2.95	Destabilizing	0.93	Ambiguous	-6.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.75	Pathogenic	0.04	Affected	3.37	25	-3	-2	0.5	-76.10	200.1	77.8	0.0	0.0	-0.2	0.1								Potentially Pathogenic	The phenol ring of Tyr342, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), faces outward in the C2 domain. In the WT simulations, the phenol ring of Tyr342 contributes to a triple tyrosine stack (Tyr342, Tyr328, and Tyr281) that links together three anti-parallel β sheet strands. Additionally, it shields Gly344 from the solvent, reducing its exposure and providing stability for the β-sandwich. This motif also contributes to a twist formation in the β sheet.In the variant simulations, the Ser342 side chain cannot participate in the stack formation. Instead, the hydroxyl group of the Ser342 side chain forms a hydrogen bond with the imidazole ring of His326 in a neighboring β strand (res. Ala322-Asp330). This disrupts the formation of a hydrogen bond between His326 and the carboxylate group of the Glu283 side chain from another β strand (res. Arg279-Cys285). Although these changes in surface interactions could weaken the characteristic twist that strengthens the β sheet fold, no major structural effects are observed in the variant simulations. The residue swap could also affect the SynGAP-membrane association, as the hydroxyl group of Ser342 could form hydrogen bonds with membrane-facing loop residues. However, this phenomenon cannot be addressed using solvent-only simulations.
c.1714T>G	W572G (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-17.692	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.900	Likely Pathogenic	6.57	Destabilizing	0.2	7.57	Destabilizing	7.07	Destabilizing	1.83	Destabilizing	-11.98	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.24	Pathogenic	0.00	Affected	3.37	35	-7	-2	0.5	-129.16	195.2	127.9	0.0	0.0	-1.0	0.0							X	Potentially Pathogenic	The introduced residue Gly572, located in an α-helix (res. Arg563-Glu578), is considerably smaller than the tryptophan it replaced. The indole ring of the Trp572 side chain lies in a hydrophobic inter-helix space, where it makes extensive hydrophobic interactions with nearby residues such as Met470, Phe569, Leu588, and Ile589. In the variant simulations, all these favorable packing interactions are completely removed, as the introduced residue Gly572 essentially lacks a side chain altogether. Although not observed in the simulations, the residue swap could also weaken the integrity of the helix (res. Arg563-Glu578), as glycine is known as an “α-helix breaker.” Overall, the residue swap is highly likely to cause critical protein folding problems that are underestimated based on the effects seen in the variant simulations.
c.218G>A	R73K	Likely Benign		Uncertain		1	6-33425826-G-A	2	1.24e-6	-4.033	Likely Benign	0.151	Likely Benign	Likely Benign	0.077	Likely Benign										-0.46	Neutral	0.053	Benign	0.007	Benign	4.14	Benign	0.00	Affected	4.32	1	2	3	0.6	-28.01
c.2195G>A	R732K	Likely Benign		Conflicting		2	6-33441660-G-A	4	2.48e-6	-5.278	Likely Benign	0.240	Likely Benign	Likely Benign	0.045	Likely Benign										-0.82	Neutral	0.973	Probably Damaging	0.943	Probably Damaging	2.69	Benign	0.21	Tolerated	3.59	7	3	2	0.6	-28.01
c.3209G>A	R1070K	Likely Benign		Conflicting		2				-5.093	Likely Benign	0.326	Likely Benign	Likely Benign	0.104	Likely Benign										-1.42	Neutral	0.049	Benign	0.048	Benign	3.86	Benign	0.09	Tolerated	3.77	5	3	2	0.6	-28.01
c.3511_3512delinsTG	A1171C	Likely Benign	Coiled-coil	Uncertain		1				-5.363	Likely Benign	0.496	Ambiguous	Likely Benign												-1.16	Neutral	0.978	Probably Damaging	0.825	Possibly Damaging	5.32	Benign	0.02	Affected	4.32	4	-2	0	0.7	32.06
c.1045C>T	P349S (3D Viewer)		C2	Uncertain		1				-7.654	In-Between	0.217	Likely Benign	Likely Benign	0.277	Likely Benign	1.92	Ambiguous	0.1	2.28	Destabilizing	2.10	Destabilizing	0.87	Ambiguous	-6.13	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	1.66	Pathogenic	0.06	Tolerated	3.37	25	1	-1	0.8	-10.04	194.9	-18.1	-0.1	0.0	0.2	0.1	X						X	Potentially Pathogenic	The cyclic pyrrolidine side chain of Pro349, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), allows the strand to end and make a tight turn before a short α helical section within a loop connecting to another β strand (res. Thr359-Pro364). In the variant simulations, the hydroxyl group of Ser349 forms a hydrogen bond with the backbone amide group of Ala351 in the short helical section. Conversely, the backbone amide group of Ser349 (absent in proline) does not form any intra-protein hydrogen bonds. However, the β strand end connects to the α helical section in a more stable and consistent manner compared to the WT. Although the residue swap does not cause major adverse effects on the protein structure in the simulations, it is possible that the tight turn at the β strand end could not be created during folding without the presence of proline.
c.136C>T	P46S	Likely Benign		Uncertain		1				-3.338	Likely Benign	0.302	Likely Benign	Likely Benign	0.066	Likely Benign										-0.60	Neutral	0.909	Possibly Damaging	0.901	Possibly Damaging	4.15	Benign	0.00	Affected			1	-1	0.8	-10.04
c.196C>T	P66S	Likely Benign		Benign		1	6-33425804-C-T	2	1.24e-6	-2.760	Likely Benign	0.929	Likely Pathogenic	Ambiguous	0.081	Likely Benign										-1.69	Neutral	0.909	Possibly Damaging	0.641	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
c.2101C>T	P701S (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441360-C-T	3	1.86e-6	-4.375	Likely Benign	0.221	Likely Benign	Likely Benign	0.132	Likely Benign	1.33	Ambiguous	0.0	0.12	Likely Benign	0.73	Ambiguous	-0.36	Likely Benign	0.78	Neutral	0.044	Benign	0.025	Benign	3.48	Benign	1.00	Tolerated	3.47	10	-1	1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.2200C>T	P734S	Likely Benign		Uncertain		2	6-33441665-C-T	2	1.24e-6	-4.291	Likely Benign	0.077	Likely Benign	Likely Benign	0.030	Likely Benign										-2.44	Neutral	0.344	Benign	0.048	Benign	2.77	Benign	0.11	Tolerated	3.64	6	1	-1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.2221C>T	P741S	Likely Benign		Uncertain		2	6-33441686-C-T	3	1.86e-6	-3.700	Likely Benign	0.063	Likely Benign	Likely Benign	0.076	Likely Benign										-0.27	Neutral	0.270	Benign	0.136	Benign	2.92	Benign	0.00	Affected	4.32	2	1	-1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.1813C>T	P605S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.830	Likely Pathogenic	0.987	Likely Pathogenic	Likely Pathogenic	0.718	Likely Pathogenic	3.40	Destabilizing	0.1	3.34	Destabilizing	3.37	Destabilizing	1.00	Destabilizing	-7.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	0.70	Pathogenic	0.00	Affected	3.37	35	1	-1	0.8	-10.04	213.8	-15.4	-0.3	0.2	0.2	0.1				X			X	Potentially Pathogenic	Pro605 is located in a short turn between an α helix (res. Glu582-Met603) and a short α helical section (res. Ser606-Phe608). The pyrrolidine side chain of Pro605 packs hydrophobically with nearby hydrophobic residues (e.g., Ile514, Leu623, Leu610) in the inter-helix space. Additionally, proline lacks a free backbone amide group, which breaks the α helix and facilitates the turn in the WT structure.In the variant simulations, the hydroxyl side chain of Ser605 forms hydrogen bonds with the backbone carbonyl groups of Ala601 and Ile602. Importantly, the helix end is more stable than with Pro605 in the WT. Indeed, proline is a more effective secondary structure breaker compared to serine.Thus, the residue swap could have a more profound effect on the actual folding process, for example, by preventing the bending at the α helix end, than what the simulations suggest. Moreover, due to its location at the GAP-Ras interface, the residue swap could affect the GAP-Ras association.
c.2359C>T	P787S		SH3-binding motif	Uncertain		1	6-33442911-C-T	3	1.86e-6	-4.203	Likely Benign	0.564	Ambiguous	Likely Benign	0.221	Likely Benign										-3.81	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.48	Pathogenic	0.02	Affected	3.64	6	-1	1	0.8	-10.04
c.2434C>T	P812S	Likely Benign	SH3-binding motif	Uncertain		1	6-33442986-C-T	1	6.20e-7	-5.689	Likely Benign	0.456	Ambiguous	Likely Benign	0.162	Likely Benign										-0.62	Neutral	0.999	Probably Damaging	0.966	Probably Damaging	2.89	Benign	0.95	Tolerated	4.32	4	1	-1	0.8	-10.04
c.2143C>T	P715S (3D Viewer)		GAP	Likely Pathogenic		1	6-33441608-C-T	1	6.20e-7	-7.635	In-Between	0.787	Likely Pathogenic	Ambiguous	0.277	Likely Benign	3.54	Destabilizing	0.0	0.81	Ambiguous	2.18	Destabilizing	0.94	Ambiguous	-7.17	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.43	Benign	0.01	Affected	3.50	9	1	-1	0.8	-10.04	231.8	-14.0	-0.1	0.0	-0.8	0.1							X	Uncertain	Pro715, along with Gly712 and Pro713, are located in a hinge region of an α-helix making a ~90-degree turn (res. Lys705-Leu725). In the WT simulations, the pyrrolidine side chain of Pro715, lacking the backbone amide groups altogether, forces the tight helix turn to take place while also hydrophobically packing with nearby residues (e.g., Leu700, Leu708, Leu714, and Leu718). Leu715, with a normal amide backbone, could potentially affect protein folding and turn formation, although this was not observed in the variant simulations. Additionally, the hydroxyl group of the Ser715 side chain can form hydrogen bonds with the backbone carbonyl group of Gly712 and disrupt the hydrophobic packing arrangement of the leucine residues from the neighboring α-helices, impacting the GAP domain tertiary assembly.
c.280C>T	P94S	Likely Benign		Benign		1	6-33425888-C-T	5	3.10e-6	-3.151	Likely Benign	0.084	Likely Benign	Likely Benign	0.093	Likely Benign										-2.36	Neutral	0.092	Benign	0.008	Benign	4.13	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04