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.2752G>A	A918T	Likely Benign		Uncertain		1	6-33443304-G-A	1	6.20e-7	-4.139	Likely Benign	0.083	Likely Benign	Likely Benign	0.065	Likely Benign										-1.09	Neutral	0.980	Probably Damaging	0.721	Possibly Damaging	2.64	Benign	0.03	Affected	4.32	4	0	1	-2.5	30.03
c.2753C>T	A918V	Likely Benign		Uncertain		3	6-33443305-C-T	2	1.24e-6	-3.684	Likely Benign	0.112	Likely Benign	Likely Benign	0.119	Likely Benign										-1.61	Neutral	0.980	Probably Damaging	0.782	Possibly Damaging	2.61	Benign	0.03	Affected	4.32	4	0	0	2.4	28.05
c.3260C>T	S1087F			Uncertain		1				-3.843	Likely Benign	0.497	Ambiguous	Likely Benign	0.105	Likely Benign										-2.75	Deleterious	0.990	Probably Damaging	0.796	Possibly Damaging	2.56	Benign	0.03	Affected	3.77	5	-2	-3	3.6	60.10
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.3377G>T	G1126V	Likely Benign		Uncertain		1	6-33443929-G-T			-6.536	Likely Benign	0.089	Likely Benign	Likely Benign	0.357	Likely Benign										-1.20	Neutral	0.009	Benign	0.008	Benign	4.76	Benign	0.03	Affected	3.77	5	-1	-3	4.6	42.08
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.371C>T	A124V	Likely Benign		Conflicting		2	6-33432236-C-T	9	5.58e-6	-4.259	Likely Benign	0.138	Likely Benign	Likely Benign	0.073	Likely Benign										-1.52	Neutral	0.173	Benign	0.009	Benign	4.07	Benign	0.03	Affected	3.61	5	0	0	2.4	28.05
c.3824G>A	R1275Q	Likely Benign		Uncertain		1	6-33447872-G-A	2	1.29e-6	-4.928	Likely Benign	0.121	Likely Benign	Likely Benign	0.103	Likely Benign										-1.72	Neutral	0.898	Possibly Damaging	0.147	Benign	2.59	Benign	0.03	Affected	3.77	5	1	1	1.0	-28.06
c.3920C>A	P1307Q	Likely Benign		Uncertain		1	6-33451794-C-A			-4.227	Likely Benign	0.114	Likely Benign	Likely Benign	0.192	Likely Benign										-0.88	Neutral	0.988	Probably Damaging	0.765	Possibly Damaging	2.82	Benign	0.03	Affected	3.77	5	0	-1	-1.9	31.01
c.3983G>A	R1328Q	Likely Benign		Uncertain		3	6-33451857-G-A	35	1.49e-4	-2.921	Likely Benign	0.273	Likely Benign	Likely Benign	0.043	Likely Benign										-1.02	Neutral	0.799	Possibly Damaging	0.098	Benign	4.12	Benign	0.03	Affected	3.77	5	1	1	1.0	-28.06
c.4021G>A	A1341T	Likely Benign		Conflicting		3	6-33451895-G-A	45	3.44e-5	-3.224	Likely Benign	0.081	Likely Benign	Likely Benign	0.099	Likely Benign										-0.58	Neutral	0.000	Benign	0.000	Benign	4.09	Benign	0.03	Affected	3.77	5	1	0	-2.5	30.03
c.707C>T	A236V (3D Viewer)		PH	Benign/Likely benign		2	6-33435558-C-T	6	3.72e-6	-8.752	Likely Pathogenic	0.267	Likely Benign	Likely Benign	0.777	Likely Pathogenic	0.61	Ambiguous	0.2	1.08	Ambiguous	0.85	Ambiguous	0.64	Ambiguous	-3.55	Deleterious	0.981	Probably Damaging	0.446	Benign	5.79	Benign	0.03	Affected	3.40	14	0	0	2.4	28.05	213.8	-44.7	0.0	0.0	-0.2	0.2						X		Potentially Benign	The methyl side chain of Ala236, located on an α helix (residues Ala236-Val250) facing an anti-parallel β sheet strand (residues Ile205-Val209), interacts hydrophobically with nearby residues such as Arg239 and Phe218. In the variant simulations, the isopropyl branched hydrocarbon side chain of Val236 maintains similar hydrophobic interactions as alanine in the WT, with an overall arrangement remarkably similar to Ala236. The residue swap does not affect the protein structure based on the simulations.
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.815G>A	R272Q (3D Viewer)		C2	Uncertain		2	6-33437720-G-A	14	8.67e-6	-9.559	Likely Pathogenic	0.286	Likely Benign	Likely Benign	0.321	Likely Benign	0.73	Ambiguous	0.1	0.15	Likely Benign	0.44	Likely Benign	1.00	Destabilizing	-1.81	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	1.88	Pathogenic	0.03	Affected	3.38	19	1	1	1.0	-28.06	255.7	52.9	0.0	0.0	-0.2	0.1				X				Uncertain	The guanidinium group of Arg272, located at the end of an anti-parallel β sheet strand (res. Arg259-Arg272), is stably maintained in an upright and outward position via stacking with the indole ring of the Trp362 side chain in another β strand (res. Thr359-Pro364). In the WT simulations, Arg272 forms hydrogen bonds with the glycine-rich Ω loop residues (res. Val365-Pro398, e.g., Gly380) and creates a salt bridge with the carboxylate group of the Asp304 side chain.In the variant simulations, the carboxamide group of the Gln272 side chain does not stack with the indole ring of Trp362 as stably as the guanidinium group of Arg272 in the WT. Consequently, the Gln272 side chain is freer to interact with the loop residues than Arg272, potentially negatively affecting the dynamic SynGAP-membrane association. Additionally, Arg272 faces the RasGTPase interface, so the residue swap could impact the SynGAP-Ras complex formation and GTPase activation.
c.844T>A	C282S (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-11.846	Likely Pathogenic	0.958	Likely Pathogenic	Likely Pathogenic	0.460	Likely Benign	1.55	Ambiguous	0.1	1.23	Ambiguous	1.39	Ambiguous	1.62	Destabilizing	-9.19	Deleterious	0.997	Probably Damaging	0.994	Probably Damaging	1.64	Pathogenic	0.03	Affected	3.39	18	0	-1	-3.3	-16.06	233.2	14.8	-0.1	0.0	-0.2	0.3						X		Potentially Benign	The thiol-containing side chain of Cys282, located at the beginning of an anti-parallel β sheet strand (res. Arg279-Leu286), packs against multiple hydrophobic residues (e.g., Ile268, Leu284, Trp308, Leu327). In the variant simulations, the hydroxyl-containing side chain of Ser282 is more hydrophilic and, hence, not as favorable as Cys282 for this hydrophobic niche. Due to this polarity difference, the residue swap could potentially weaken the hydrophobic packing of the C2 domain during the folding process.Moreover, because the C2 domain interacts with the membrane, there could also be a negative effect on the stability of the SynGAP-membrane association. However, no large-scale structural changes were observed during the variant simulations. The hydroxyl group of Ser282 forms a hydrogen bond with the backbone carbonyl group of His326 in another β strand (res. Ala322-Arg329), which competes directly with the backbone amide group of Glu283 within the secondary structure element.
c.953C>T	P318L (3D Viewer)	Likely Pathogenic	C2	Uncertain		3	6-33437858-C-T	3	1.86e-6	-10.090	Likely Pathogenic	0.958	Likely Pathogenic	Likely Pathogenic	0.624	Likely Pathogenic	1.33	Ambiguous	0.1	0.26	Likely Benign	0.80	Ambiguous	0.43	Likely Benign	-8.96	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.82	Pathogenic	0.03	Affected	3.38	23	-3	-3	5.4	16.04	228.6	-68.9	-0.7	0.7	-0.4	0.1						X		Potentially Benign	The cyclic five-membered pyrrolidine ring of Pro318, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Asp330-Ala322, res. Thr305-Asn315), packs against the hydrophobic side chain of Ile205 at the end of the anti-parallel β sheet in the PH domain. In the variant simulations, the iso-butyl side chain of Leu318 is unable to do the same, potentially weakening the PH and C2 domain association. Importantly, the residue swap could also affect loop formation during folding, as proline can make tighter turns than leucine. Because the residue swap could affect the C2 domain stability, it could also negatively impact the SynGAP-membrane association.
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.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.1202G>A	R401Q (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33438107-G-A			-11.213	Likely Pathogenic	0.969	Likely Pathogenic	Likely Pathogenic	0.780	Likely Pathogenic	0.96	Ambiguous	0.1	1.50	Ambiguous	1.23	Ambiguous	1.20	Destabilizing	-3.69	Deleterious	0.999	Probably Damaging	0.978	Probably Damaging	5.47	Benign	0.04	Affected	3.38	27	1	1	1.0	-28.06
c.1136C>T	S379L (3D Viewer)	Likely Benign	C2	Benign		1	6-33438041-C-T	8	4.05e-5	-5.641	Likely Benign	0.173	Likely Benign	Likely Benign	0.469	Likely Benign	0.39	Likely Benign	0.2	3.38	Destabilizing	1.89	Ambiguous	-0.52	Ambiguous	-0.85	Neutral	0.015	Benign	0.002	Benign	3.83	Benign	0.04	Affected	4.32	11	-3	-2	4.6	26.08	251.9	-48.1	0.6	1.1	0.0	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 leucine are rarely tolerated. Although no negative structural effects are observed in the variant simulations, Leu379 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.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.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.1390T>G	F464V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.254	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.592	Likely Pathogenic	3.61	Destabilizing	0.1	2.89	Destabilizing	3.25	Destabilizing	1.40	Destabilizing	-6.96	Deleterious	0.998	Probably Damaging	0.996	Probably Damaging	3.36	Benign	0.04	Affected	3.37	34	-1	-1	1.4	-48.04	210.1	40.5	-0.1	0.0	-0.9	0.3							X	Potentially Pathogenic	The phenyl ring of Phe464, located in the middle of an α helix (res. Ala461–Phe476), packs against hydrophobic residues (e.g., Met468, Leu451, Leu455, and Tyr428) in the inter-helix space formed with two other α helices (res. Asn440-Lys460 and res. Pro413-Glu436). The iso-propyl side chain of Val464 is similarly hydrophobic but considerably smaller than the original phenyl ring of Phe464. To compensate for the size difference, neighboring residues need to fill in the gap in the variant simulations.The phenolic side chain of Tyr428, located at the middle bend of an α helix (res. Glu436-Pro413), assumes a new position in the inter-helix space or rotates inward next to the third α helix (res. Asn440-Lys460) when the stable H-bond between Tyr428 and Asp467 seen in the WT simulations breaks. The residue swap also leads to the loss of the methionine-aromatic interaction between the Met468 and Phe464 side chains, which could weaken the integrity of the parent α helix (res. Ala461-Phe476). Although the simulations likely underestimate the full adverse effect of the introduced mutation during folding, the two opposing α helices (res. Ala461–Phe476 and res. Glu436-Pro413) move substantially closer to each other in the variant simulations.
c.1403T>A	M468K (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-16.982	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.828	Likely Pathogenic	3.21	Destabilizing	0.1	3.30	Destabilizing	3.26	Destabilizing	2.57	Destabilizing	-4.61	Deleterious	0.878	Possibly Damaging	0.922	Probably Damaging	-1.34	Pathogenic	0.04	Affected	3.37	31	0	-1	-5.8	-3.02	188.7	69.3	0.0	0.0	-0.1	0.2			X				X	Potentially Pathogenic	The thioether group of Met468, located in the middle of an α helix (res. Ala461–Phe476), interacts with hydrophobic residues (e.g., Phe464, Leu465, Leu489) in an inter-helix space formed by two other α helices (res. Ala461–Phe476, res. Thr488–Gly502). In the variant simulations, the positively charged side chain of Lys468 rotates outward to escape the hydrophobic niche, forming an H-bond with the hydroxyl group of the Ser471 side chain and a salt bridge with the carboxylate group of the Glu472 side chain. This residue swap also disrupts the methionine-aromatic stacking with the phenyl ring of the Phe464 side chain. Although no large-scale structural changes are observed during the variant simulations, the importance of hydrophobic packing suggests that the effects could be more pronounced during protein folding.
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.1712C>T	S571L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440764-C-T	1	6.23e-7	-11.651	Likely Pathogenic	0.660	Likely Pathogenic	Likely Benign	0.841	Likely Pathogenic	-1.53	Ambiguous	0.1	-1.05	Ambiguous	-1.29	Ambiguous	0.27	Likely Benign	-5.61	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	-1.25	Pathogenic	0.04	Affected	3.37	35	-2	-3	4.6	26.08
c.2029A>T	S677C (3D Viewer)	Likely Benign	GAP	Benign		1				-8.496	Likely Pathogenic	0.076	Likely Benign	Likely Benign	0.153	Likely Benign	-0.51	Ambiguous	0.3	-0.30	Likely Benign	-0.41	Likely Benign	0.15	Likely Benign	-2.41	Neutral	0.932	Possibly Damaging	0.222	Benign	3.25	Benign	0.04	Affected	3.41	23	-1	0	3.3	16.06
c.2210A>C	Q737P	Likely Benign		Uncertain		1				-2.407	Likely Benign	0.054	Likely Benign	Likely Benign	0.154	Likely Benign										-1.22	Neutral	0.005	Benign	0.013	Benign	2.78	Benign	0.04	Affected	4.07	3	-1	0	1.9	-31.01
c.2561G>A	R854H	Likely Benign		Uncertain		1	6-33443113-G-A	4	2.48e-6	-3.686	Likely Benign	0.094	Likely Benign	Likely Benign	0.183	Likely Benign										-1.38	Neutral	0.997	Probably Damaging	0.899	Possibly Damaging	4.07	Benign	0.04	Affected	3.88	3	2	0	1.3	-19.05
c.2668C>T	R890C			Benign		1	6-33443220-C-T	9	5.58e-6	-5.786	Likely Benign	0.402	Ambiguous	Likely Benign	0.200	Likely Benign										-3.38	Deleterious	1.000	Probably Damaging	0.971	Probably Damaging	3.94	Benign	0.04	Affected	4.32	4	-4	-3	7.0	-53.05
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.3449C>T	A1150V	Likely Benign		Uncertain		1	6-33444484-C-T	3	1.86e-6	-3.648	Likely Benign	0.192	Likely Benign	Likely Benign	0.066	Likely Benign										-2.22	Neutral	0.114	Benign	0.055	Benign	2.32	Pathogenic	0.04	Affected	3.77	5	0	0	2.4	28.05
c.3557C>T	S1186L		Coiled-coil	Uncertain		1	6-33444592-C-T			-4.829	Likely Benign	0.923	Likely Pathogenic	Ambiguous	0.177	Likely Benign										-2.58	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	2.65	Benign	0.04	Affected	3.82	4	-3	-2	4.6	26.08
c.3705G>A	M1235I	Likely Benign	Coiled-coil	Uncertain		1				-4.312	Likely Benign	0.310	Likely Benign	Likely Benign	0.027	Likely Benign										-1.44	Neutral	0.139	Benign	0.056	Benign	2.69	Benign	0.04	Affected	3.77	5	1	2	2.6	-18.03
c.3859C>A	P1287T	Likely Benign		Uncertain		1	6-33447907-C-A			-3.940	Likely Benign	0.077	Likely Benign	Likely Benign	0.044	Likely Benign										-0.22	Neutral	0.126	Benign	0.041	Benign	2.78	Benign	0.04	Affected	3.77	5	-1	0	0.9	3.99
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.3920C>T	P1307L	Likely Benign		Benign		1	6-33451794-C-T	11	6.82e-6	-4.044	Likely Benign	0.144	Likely Benign	Likely Benign	0.292	Likely Benign										-1.49	Neutral	0.779	Possibly Damaging	0.220	Benign	2.82	Benign	0.04	Affected	3.77	5	-3	-3	5.4	16.04
c.515G>A	R172Q			Uncertain		1	6-33435157-G-A	3	1.86e-6	-7.245	In-Between	0.465	Ambiguous	Likely Benign	0.135	Likely Benign										-1.72	Neutral	0.804	Possibly Damaging	0.091	Benign	4.04	Benign	0.04	Affected	3.61	5	1	1	1.0	-28.06
c.583G>C	A195P	Likely Pathogenic		Likely Pathogenic		1				-9.715	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.152	Likely Benign										-3.03	Deleterious	0.997	Probably Damaging	0.916	Probably Damaging	4.00	Benign	0.04	Affected	3.54	6	1	-1	-3.4	26.04
c.878G>A	R293H	Likely Pathogenic	C2	Uncertain		1				-13.009	Likely Pathogenic	0.973	Likely Pathogenic	Likely Pathogenic	0.438	Likely Benign	4.45	Destabilizing	2.3	2.12	Destabilizing	3.29	Destabilizing	0.32	Likely Benign	-4.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.45	Pathogenic	0.04	Affected			2	0	1.3	-19.05
c.1082A>C	Q361P (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-13.280	Likely Pathogenic	0.956	Likely Pathogenic	Likely Pathogenic	0.482	Likely Benign	3.12	Destabilizing	0.0	3.45	Destabilizing	3.29	Destabilizing	0.38	Likely Benign	-3.03	Deleterious	0.996	Probably Damaging	0.979	Probably Damaging	1.63	Pathogenic	0.05	Affected	3.37	25	-1	0	1.9	-31.01
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.1169G>A	G390E (3D Viewer)		C2	Uncertain		1				-7.913	In-Between	0.646	Likely Pathogenic	Likely Benign	0.575	Likely Pathogenic	2.61	Destabilizing	0.9	4.28	Destabilizing	3.45	Destabilizing	0.47	Likely Benign	-0.87	Neutral	0.276	Benign	0.045	Benign	1.32	Pathogenic	0.05	Affected	4.32	8	0	-2	-3.1	72.06	241.5	-108.4	0.6	0.5	-0.1	0.1								Uncertain	Gly390 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364 and res. Ala399-Ile411). The Ω loop is assumed to directly interact with the membrane, and it is observed to move arbitrarily throughout the WT solvent simulations. This 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 significant 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 Glu390 may not be as well tolerated in the Ω loop. Additionally, the carboxylate group of Glu390 occasionally forms H-bonds with other loop residues in the variant simulations. The interaction between the acidic carboxylate side chain and the acidic membrane lipids may further influence the SynGAP-membrane complex. However, since the effects on the Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
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.1292T>C	L431P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-14.222	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.659	Likely Pathogenic	6.78	Destabilizing	0.3	11.59	Destabilizing	9.19	Destabilizing	2.29	Destabilizing	-6.39	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.91	Benign	0.05	Affected	3.37	29	-3	-3	-5.4	-16.04	222.4	62.8	0.1	0.0	0.1	0.0	X							Potentially Pathogenic	The iso-butyl side chain of Leu431, located in an α helix (res. Met414-Glu436), packs against other hydrophobic residues in an interhelix space (e.g., Val434, Leu435, Leu696, Leu711) in the WT simulations. While the backbone amide group of Leu431 forms an H-bond with the carbonyl group of His427, the cyclic five-membered pyrrolidine ring of Pro431, lacking the necessary amide group, cannot do the same. Thus, although the cyclic five-membered pyrrolidine ring of Pro431 packs almost as favorably as the side chain of Leu431 in the hydrophobic niche, the residue swap causes the α helix to partially unfold in the variant simulations.
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.1631G>C	R544P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2				-16.905	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.762	Likely Pathogenic	4.70	Destabilizing	0.1	4.19	Destabilizing	4.45	Destabilizing	1.14	Destabilizing	-4.88	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.48	Pathogenic	0.05	Affected	3.37	35	0	-2	2.9	-59.07	192.0	123.8	0.1	0.0	-0.3	0.0	X	X						Potentially Pathogenic	Arg544 is located in the middle of an α-helix (res. Ala533-Val560). In the WT simulations, the guanidinium side chain of Arg544 forms a salt bridge with the carboxylate groups of Glu548 on the same α-helix, and with Glu651 and Glu656 on an opposing α-helix (res. Glu666-Asp644). In the variant simulations, the pyrrolidine side chain of Pro544 cannot form any of the salt bridges that Arg544 does in the WT, potentially weakening the tertiary structure assembly. Additionally, Pro544 lacks the amide group, and thus, unlike Arg544 in the WT, is unable to form a hydrogen bond with the carbonyl of Gln540. This disruption breaks the continuity of the secondary structure element, causing the α-helix to bend slightly in the variant simulations. These negative structural effects could be more pronounced during protein folding and are likely to be undermined in the MD simulations.
c.1976C>T	S659F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.925	Likely Pathogenic	0.662	Likely Pathogenic	Likely Benign	0.194	Likely Benign	-0.81	Ambiguous	0.1	-0.25	Likely Benign	-0.53	Ambiguous	0.32	Likely Benign	-4.59	Deleterious	0.806	Possibly Damaging	0.171	Benign	3.39	Benign	0.05	Affected	3.38	28	-3	-2	3.6	60.10	221.3	-61.2	0.0	0.0	0.6	0.4						X		Potentially Benign	In the WT simulations, the hydroxyl group of Ser659, located in a kink in the middle of the long α-helix (res. Ser641-Glu666), forms a hydrogen bond with the carboxylate group of Glu656. However, the phenol ring of the Phe659 side chain cannot form a similar hydrogen bond. Instead, it interacts with the hydrophobic isopropyl side chain of Val555 from the opposing α-helix (res. Ala533-Val560). This residue swap may therefore cause issues during protein folding.
c.1724G>A	R575H (3D Viewer)		GAP	Conflicting		4	6-33440776-G-A	204	1.27e-4	-11.142	Likely Pathogenic	0.496	Ambiguous	Likely Benign	0.707	Likely Pathogenic	0.81	Ambiguous	0.2	-0.22	Likely Benign	0.30	Likely Benign	1.31	Destabilizing	-2.34	Neutral	1.000	Probably Damaging	0.998	Probably Damaging	-1.33	Pathogenic	0.05	Affected	3.37	35	2	0	1.3	-19.05	244.7	80.6	0.0	0.0	0.3	0.0		X						Potentially Pathogenic	The guanidinium group of Arg575, located in an α-helix (res. Arg563-Glu578), forms salt bridges with the carboxylate groups of Asp463 and Asp467, and it also hydrogen bonds with the hydroxyl group of Ser466 on an opposing α-helix (res. Ala461-Phe476) in the WT simulations. In the variant simulations, the imidazole ring of His575 (in its neutral epsilon protonated form) cannot form the same salt bridges as the guanidinium group of the non-mutated Arg575. Instead, His575 only forms weak hydrogen bonds with the hydroxyl groups of Ser466 and Ser571. Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.
c.1904A>G	N635S (3D Viewer)		GAP	Conflicting		4	6-33440956-A-G	10	6.20e-6	-9.002	Likely Pathogenic	0.101	Likely Benign	Likely Benign	0.104	Likely Benign	0.80	Ambiguous	0.1	0.67	Ambiguous	0.74	Ambiguous	0.95	Ambiguous	-4.45	Deleterious	0.261	Benign	0.044	Benign	3.06	Benign	0.05	Affected	3.37	34	1	1	2.7	-27.03	196.0	30.9	0.1	0.0	-0.3	0.2				X				Uncertain	In the WT simulations, the carboxamide side chain of Asn635, located on the outer surface of an α helix (res. Glu617-Asn635), forms hydrogen bonds with Gln631 on the same α helix and with the hydroxyl side chain of Ser590 on an opposing α helix (res. Glu582-Met603).In the variant simulations, the side chain of Ser635 is shorter than asparagine and thus prefers to hydrogen bond with the carbonyl group of Gln631 on the same helix and, to a lesser extent, with Ser590 compared to Asn635 in the WT. Ser635 forms hydrogen bonds with the backbone atoms of the same helix, which may destabilize the helix, although this is not clearly evident in the simulations. The weakening of the hydrogen bond between Ser635 and Ser590 in the variant may also weaken the tertiary structure assembly between the helices.Additionally, Asn635 is at the GTPase interface. However, the implication of the residue swap on the complex formation with the GTPase cannot be investigated using solvent-only simulations.
c.2627C>T	S876L			Uncertain		2				-5.856	Likely Benign	0.489	Ambiguous	Likely Benign	0.249	Likely Benign										-3.56	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	2.57	Benign	0.05	Affected	3.77	5	-2	-3	4.6	26.08
c.2650C>T	R884W	Likely Benign		Uncertain		1	6-33443202-C-T	5	3.10e-6	-3.785	Likely Benign	0.332	Likely Benign	Likely Benign	0.151	Likely Benign										0.26	Neutral	0.995	Probably Damaging	0.812	Possibly Damaging	2.56	Benign	0.05	Affected	4.32	4	-3	2	3.6	30.03
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.2914C>T	P972S	Likely Benign		Uncertain		1	6-33443466-C-T	4	2.48e-6	-4.008	Likely Benign	0.058	Likely Benign	Likely Benign	0.074	Likely Benign										-0.38	Neutral	0.001	Benign	0.002	Benign	4.28	Benign	0.05	Affected	4.32	2	-1	1	0.8	-10.04
c.3038C>G	S1013C	Likely Benign		Uncertain		1	6-33443590-C-G	4	2.48e-6	-6.745	Likely Benign	0.110	Likely Benign	Likely Benign	0.058	Likely Benign										-2.06	Neutral	0.898	Possibly Damaging	0.579	Possibly Damaging	2.64	Benign	0.05	Affected	3.77	5	0	-1	3.3	16.06
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.3290C>T	P1097L	Likely Benign		Benign		1				-4.410	Likely Benign	0.145	Likely Benign	Likely Benign	0.131	Likely Benign										-2.07	Neutral	0.611	Possibly Damaging	0.198	Benign	2.64	Benign	0.05	Affected	3.77	5	-3	-3	5.4	16.04
c.3354C>A	S1118R	Likely Benign		Uncertain		1				-2.670	Likely Benign	0.553	Ambiguous	Likely Benign	0.166	Likely Benign										-0.74	Neutral	0.034	Benign	0.023	Benign	5.17	Benign	0.05	Affected	4.32	2	-1	0	-3.7	69.11
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.3941C>T	P1314L	Likely Benign		Likely Benign		1	6-33451815-C-T	2	1.24e-6	-4.040	Likely Benign	0.118	Likely Benign	Likely Benign	0.049	Likely Benign										-0.20	Neutral	0.421	Benign	0.066	Benign	4.19	Benign	0.05	Affected	3.77	5	-3	-3	5.4	16.04
c.611C>G	S204C (3D Viewer)	Likely Benign	PH	Uncertain		1				-6.613	Likely Benign	0.127	Likely Benign	Likely Benign	0.148	Likely Benign	0.65	Ambiguous	0.4	-1.13	Ambiguous	-0.24	Likely Benign	0.10	Likely Benign	-0.64	Neutral	0.978	Probably Damaging	0.753	Possibly Damaging	4.13	Benign	0.05	Affected	3.44	10	0	-1	3.3	16.06	223.6	-13.8	0.6	0.3	0.0	0.2	X							Uncertain	The hydroxyl-containing Ser204, located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by the thiol-containing cysteine. In the WT simulations, Ser204 simultaneously forms hydrogen bonds with the backbone carbonyl of Asp201 and the hydroxyl group of Thr224, helping to stabilize the two anti-parallel β strands (res. Ile205-Lys207 and Cys219-Thr223) at the end of the β sheet. Since the thiol group of cysteine forms weaker hydrogen bonds than the hydroxyl group of serine, Cys204 does not maintain the hydrogen bond network as stably as Ser204 in the variant simulations. However, because the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
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.986G>A	R329H (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437891-G-A	2	1.24e-6	-10.154	Likely Pathogenic	0.769	Likely Pathogenic	Likely Benign	0.155	Likely Benign	2.53	Destabilizing	0.7	0.71	Ambiguous	1.62	Ambiguous	0.82	Ambiguous	-3.17	Deleterious	0.995	Probably Damaging	0.778	Possibly Damaging	4.04	Benign	0.05	Affected	3.41	15	2	0	1.3	-19.05	220.4	81.4	0.1	0.1	0.2	0.3								Uncertain	The guanidinium group of Arg329, located at the end of an anti-parallel β sheet strand (res. Ala322-Asp330), faces the negatively charged lipid bilayer surface. While the residue swap does not cause any apparent negative effects on the protein structure in the variant simulations, it could adversely affect the SynGAP-membrane association in reality. The positively charged Arg329 side chain forms hydrogen bonds with other loop residues (e.g., Ser371, Asp338) that are expected to dynamically interact with the membrane head group region. However, this phenomenon is beyond the scope of the solvent-only simulations to unravel. Notably, histidine can also be double protonated and positively charged, but this alternative protonation state was not considered in the variant simulations.
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.1370G>A	S457N	Likely Pathogenic	GAP	Uncertain		1				-10.221	Likely Pathogenic	0.949	Likely Pathogenic	Ambiguous	0.241	Likely Benign	0.19	Likely Benign	0.0	-0.22	Likely Benign	-0.02	Likely Benign	0.67	Ambiguous	-2.76	Deleterious	0.940	Possibly Damaging	0.843	Possibly Damaging	3.28	Benign	0.06	Tolerated			1	1	-2.7	27.03
c.1651C>A	L551M (3D Viewer)		GAP	Uncertain		1	6-33438894-C-A	7	4.34e-6	-9.937	Likely Pathogenic	0.480	Ambiguous	Likely Benign	0.544	Likely Pathogenic	-0.07	Likely Benign	0.1	0.13	Likely Benign	0.03	Likely Benign	0.71	Ambiguous	-0.56	Neutral	1.000	Probably Damaging	1.000	Probably Damaging	-1.48	Pathogenic	0.06	Tolerated	3.37	35	4	2	-1.9	18.03	246.5	-18.6	0.0	0.0	0.3	0.0						X		Potentially Benign	L551 is located on an α-helix (res. Ala533-Val560). The iso-butyl side chain of Leu551 hydrophobically packs with nearby hydrophobic residues such as Cys547, Phe652, Leu633, and Ile630 in the inter-helix space. In the variant simulations, the thioether side chain of Met551 can maintain similar hydrophobic interactions as Leu551 in the WT, thus causing no negative effect on the protein structure during the simulations.
c.1736G>A	R579Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440788-G-A	18	1.12e-5	-9.193	Likely Pathogenic	0.690	Likely Pathogenic	Likely Benign	0.673	Likely Pathogenic	0.65	Ambiguous	0.1	0.70	Ambiguous	0.68	Ambiguous	1.13	Destabilizing	-3.31	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.34	Pathogenic	0.06	Tolerated	3.37	34	1	1	1.0	-28.06
c.1531G>A	G511R (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-11.327	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.416	Likely Benign	1.94	Ambiguous	0.3	1.32	Ambiguous	1.63	Ambiguous	0.94	Ambiguous	-7.72	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.26	Benign	0.06	Tolerated	3.37	35	-3	-2	-4.1	99.14	279.4	-159.9	0.0	0.0	0.7	0.1			X				X	Potentially Pathogenic	Gly511 is located in an α-helix (res. Gly502-Tyr518), facing hydrophobic residues in an inter-helix space (e.g., Leu610, Ile514) in the WT simulations. In contrast, in the variant simulations, the bulkier and positively charged guanidinium side chain of Arg511 forms a salt bridge with the carboxylate group of Glu217 or hydrogen bonds with the backbone carbonyl group of Leu610. Although the residue swap introduces a third positively charged residue in close vicinity (Arg511, Lys507, Arg515), the protein structure seems to remain stable in the variant simulations. Importantly, according to ClinVar, the residue swap alters the last nucleotide of an exon and is predicted to destroy the splice donor site, resulting in aberrant splicing and pathogenic status.	10.1016/j.ajhg.2020.11.011
c.1531G>C	G511R (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-11.327	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.415	Likely Benign	1.94	Ambiguous	0.3	1.32	Ambiguous	1.63	Ambiguous	0.94	Ambiguous	-7.72	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.26	Benign	0.06	Tolerated	3.37	35	-3	-2	-4.1	99.14	279.4	-159.9	0.0	0.0	0.7	0.1			X				X	Potentially Pathogenic	Gly511 is located in an α-helix (res. Gly502-Tyr518), facing hydrophobic residues in an inter-helix space (e.g., Leu610, Ile514) in the WT simulations. In contrast, in the variant simulations, the bulkier and positively charged guanidinium side chain of Arg511 forms a salt bridge with the carboxylate group of Glu217 or hydrogen bonds with the backbone carbonyl group of Leu610. Although the residue swap introduces a third positively charged residue in close vicinity (Arg511, Lys507, Arg515), the protein structure seems to remain stable in the variant simulations. Importantly, according to ClinVar, the residue swap alters the last nucleotide of an exon and is predicted to destroy the splice donor site, resulting in aberrant splicing and pathogenic status.	10.1016/j.ajhg.2020.11.011
c.1640G>A	C547Y (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-15.871	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.874	Likely Pathogenic	8.53	Destabilizing	1.8	6.20	Destabilizing	7.37	Destabilizing	0.62	Ambiguous	-10.57	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.33	Pathogenic	0.06	Tolerated	3.37	35	0	-2	-3.8	60.04	280.1	-54.8	0.0	0.0	0.0	0.0		X	X				X	Potentially Pathogenic	Cys547 is located in an α-helix (res. Ala533-Val560). The thiol side chain of Cys547 is situated in a hydrophobic inter-helix space, where it packs hydrophobically with other residues such as Ile626, Leu551, and Phe652. Additionally, the thiol side chain of Cys weakly hydrogen bonds with the carbonyl group of Leu543 in the same α-helix. In the variant simulations, the bulkier phenol ring of Tyr547, with its polar hydroxyl group, is less suited for the hydrophobic space. Consequently, it moves outside and forms a hydrogen bond with the carbonyl group of Phe652 in the neighboring α-helix (res. Glu666-Asp644). This causes the two helices to slightly separate, negatively affecting the secondary structure integrity of the latter helix. These negative structural effects could be more pronounced during protein folding and are likely to be undermined in the MD simulations.
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.2294G>A	S765N	Likely Benign		Uncertain		1				-5.098	Likely Benign	0.378	Ambiguous	Likely Benign	0.094	Likely Benign										-0.94	Neutral	0.985	Probably Damaging	0.950	Probably Damaging	4.11	Benign	0.06	Tolerated	3.64	6	1	1	-2.7	27.03
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.2459A>G	Y820C	Likely Pathogenic		Uncertain		1				-8.797	Likely Pathogenic	0.744	Likely Pathogenic	Likely Benign	0.113	Likely Benign										-3.16	Deleterious	1.000	Probably Damaging	0.983	Probably Damaging	2.68	Benign	0.06	Tolerated	3.77	5	0	-2	3.8	-60.04
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.2619C>G	S873R			Uncertain		1	6-33443171-C-G	1	6.20e-7	-5.856	Likely Benign	0.976	Likely Pathogenic	Likely Pathogenic	0.192	Likely Benign										-2.74	Deleterious	0.997	Probably Damaging	0.995	Probably Damaging	2.67	Benign	0.06	Tolerated	3.77	5	0	-1	-3.7	69.11
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.3041G>T	G1014V	Likely Benign		Uncertain		1				-4.612	Likely Benign	0.181	Likely Benign	Likely Benign	0.053	Likely Benign										-2.47	Neutral	0.818	Possibly Damaging	0.377	Benign	2.72	Benign	0.06	Tolerated	3.77	5	-1	-3	4.6	42.08
c.3100C>G	P1034A	Likely Benign		Benign		1				-4.174	Likely Benign	0.178	Likely Benign	Likely Benign	0.060	Likely Benign										-2.44	Neutral	0.059	Benign	0.061	Benign	2.47	Pathogenic	0.06	Tolerated	3.77	5	1	-1	3.4	-26.04
c.3848C>T	P1283L	Likely Benign		Benign		1	6-33447896-C-T	32	2.06e-5	-3.740	Likely Benign	0.093	Likely Benign	Likely Benign	0.047	Likely Benign										-1.04	Neutral	0.005	Benign	0.003	Benign	2.76	Benign	0.06	Tolerated	3.77	5	-3	-3	5.4	16.04
c.3956C>G	A1319G	Likely Benign		Uncertain		2	6-33451830-C-G			-3.927	Likely Benign	0.084	Likely Benign	Likely Benign	0.128	Likely Benign										-0.74	Neutral	0.819	Possibly Damaging	0.581	Possibly Damaging	4.07	Benign	0.06	Tolerated	3.77	5	1	0	-2.2	-14.03
c.895C>T	R299C (3D Viewer)	Likely Pathogenic	C2	Conflicting		2	6-33437800-C-T	3	1.86e-6	-6.326	Likely Benign	0.572	Likely Pathogenic	Likely Benign	0.344	Likely Benign	1.85	Ambiguous	0.4	0.61	Ambiguous	1.23	Ambiguous	0.76	Ambiguous	-3.54	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.65	Pathogenic	0.06	Tolerated	3.39	19	-4	-3	7.0	-53.05	210.7	91.3	0.1	0.0	0.0	0.2	X	X						Potentially Pathogenic	The guanidinium group of Arg299, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Met289-Pro298, res. Thr305-Asn315), forms hydrogen bonds that stabilize the tight turn. In the WT simulations, the Arg299 side chain hydrogen bonds with the loop backbone carbonyl groups (e.g., Ser302, Thr305, Leu274, Gly303), the hydroxyl group of Ser300, and even forms a salt bridge with the carboxylate group of Asp304.In the variant simulations, the thiol group of the Cys299 side chain is unable to form any of these well-coordinated or strong interactions, which could affect the initial formation of the secondary hairpin loop during folding. β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant. Moreover, the positively charged Arg299 side chain faces the polar head group region of the inner leaflet membrane and could directly anchor the C2 domain to the membrane. In short, the residue swap could negatively affect both protein folding and the stability of the SynGAP-membrane association.
c.1367A>C	Q456P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.250	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.469	Likely Benign	3.68	Destabilizing	0.2	8.43	Destabilizing	6.06	Destabilizing	0.82	Ambiguous	-5.66	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	3.34	Benign	0.07	Tolerated	3.37	34	-1	0	1.9	-31.01
c.1404G>A	M468I (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438436-G-A	1	6.20e-7	-8.583	Likely Pathogenic	0.907	Likely Pathogenic	Ambiguous	0.508	Likely Pathogenic	2.53	Destabilizing	0.2	1.89	Ambiguous	2.21	Destabilizing	0.37	Likely Benign	-1.06	Neutral	0.748	Possibly Damaging	0.886	Possibly Damaging	-1.10	Pathogenic	0.07	Tolerated	3.37	31	1	2	2.6	-18.03
c.1349C>A	A450E (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.578	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.653	Likely Pathogenic	3.86	Destabilizing	0.2	5.23	Destabilizing	4.55	Destabilizing	1.59	Destabilizing	-4.67	Deleterious	0.999	Probably Damaging	0.992	Probably Damaging	3.38	Benign	0.07	Tolerated	3.37	32	0	-1	-5.3	58.04	240.1	-82.6	0.0	0.0	0.7	0.0			X				X	Potentially Pathogenic	The methyl group of Ala450, located in an α helix (res. Asn440-Thr458), packs against hydrophobic residues in the inter-helix space (e.g., Leu692). In the variant simulations, the carboxylate group of the Glu450 side chain rotates outward, away from the hydrophobic niche, where it does not form any lasting salt bridges or H-bonds. Although the residue swap does not negatively affect the protein structure based on the simulations, it is possible that the introduction of the negatively charged residue adversely affects the folding process or tertiary assembly.
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.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.1621G>C	A541P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.733	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.594	Likely Pathogenic	2.47	Destabilizing	0.3	7.26	Destabilizing	4.87	Destabilizing	0.86	Ambiguous	-3.16	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.34	Pathogenic	0.07	Tolerated	3.37	35	1	-1	-3.4	26.04	170.4	-11.2	0.1	0.0	0.1	0.0	X							Potentially Pathogenic	Ala541 is located on the outer surface of an α-helix (res. Ala533-Val560). The methyl group of Ala541 is on the surface and does not form any interactions. Proline lacks a free backbone amide group, and thus, Pro541 is unable to form a hydrogen bond with the carbonyl group of Ala537 in the variant simulations. Consequently, Pro541 disrupts the continuity of the secondary structure element, causing the α-helix to bend slightly in the variant simulations.
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.2225G>A	R742Q	Likely Benign		Uncertain		2	6-33441690-G-A	24	1.49e-5	-4.090	Likely Benign	0.068	Likely Benign	Likely Benign	0.054	Likely Benign										-0.19	Neutral	0.032	Benign	0.007	Benign	2.73	Benign	0.07	Tolerated	4.32	2	1	1	1.0	-28.06
c.2302G>T	D768Y	Likely Pathogenic		Uncertain		1	6-33442460-G-T			-9.866	Likely Pathogenic	0.824	Likely Pathogenic	Ambiguous	0.234	Likely Benign										-2.86	Deleterious	0.989	Probably Damaging	0.806	Possibly Damaging	4.01	Benign	0.07	Tolerated	3.64	6	-4	-3	2.2	48.09
c.2324G>C	R775P	Likely Benign		Benign		1				-5.072	Likely Benign	0.452	Ambiguous	Likely Benign	0.168	Likely Benign										-0.79	Neutral	0.971	Probably Damaging	0.944	Probably Damaging	4.13	Benign	0.07	Tolerated	3.64	6	-2	0	2.9	-59.07
c.2015C>A	T672K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.192	Likely Pathogenic	0.698	Likely Pathogenic	Likely Benign	0.065	Likely Benign	0.20	Likely Benign	0.5	1.21	Ambiguous	0.71	Ambiguous	0.72	Ambiguous	-4.31	Deleterious	0.745	Possibly Damaging	0.051	Benign	3.40	Benign	0.07	Tolerated	3.40	25	0	-1	-3.2	27.07	195.1	7.0	0.4	0.7	0.4	0.1		X				X		Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), 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 itself, such as Lys566, Glu666, and Asn669. In the variant simulations, Lys672 can only form a hydrogen bond with the amino group of the Lys566 side chain via its backbone carbonyl group. Consequently, it cannot maintain the Lys566-Glu666 salt bridge through hydrogen bonding. However, the amino group of Lys periodically forms a salt bridge with the carboxylate group of Glu666, which prevents a drastic disruption of the hydrogen-bond network that keeps the loop close to the helices.
c.2582C>T	S861L	Likely Benign		Uncertain		1	6-33443134-C-T	2	1.24e-6	-4.966	Likely Benign	0.219	Likely Benign	Likely Benign	0.144	Likely Benign										-2.10	Neutral	0.904	Possibly Damaging	0.355	Benign	3.93	Benign	0.07	Tolerated	4.32	3	-3	-2	4.6	26.08
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.2914C>G	P972A	Likely Benign		Uncertain		1	6-33443466-C-G	1	6.20e-7	-0.167	Likely Benign	0.045	Likely Benign	Likely Benign	0.046	Likely Benign										-0.89	Neutral	0.016	Benign	0.011	Benign	4.29	Benign	0.07	Tolerated	4.32	2	-1	1	3.4	-26.04
c.2924C>A	T975N	Likely Benign		Uncertain		1	6-33443476-C-A	1	6.20e-7	-4.671	Likely Benign	0.089	Likely Benign	Likely Benign	0.100	Likely Benign										-0.58	Neutral	0.586	Possibly Damaging	0.302	Benign	4.13	Benign	0.07	Tolerated	4.32	2	0	0	-2.8	13.00
c.2960A>G	D987G	Likely Pathogenic		Uncertain		1				-4.782	Likely Benign	0.849	Likely Pathogenic	Ambiguous	0.234	Likely Benign										-2.79	Deleterious	0.943	Possibly Damaging	0.808	Possibly Damaging	2.45	Pathogenic	0.07	Tolerated	4.32	2	1	-1	3.1	-58.04
c.3326T>C	L1109P	Likely Benign		Conflicting		2				-5.313	Likely Benign	0.120	Likely Benign	Likely Benign	0.151	Likely Benign										-0.52	Neutral	0.002	Benign	0.003	Benign	2.65	Benign	0.07	Tolerated	4.32	2	-3	-3	-5.4	-16.04
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.3511G>A	A1171T	Likely Benign	Coiled-coil	Uncertain		1				-3.658	Likely Benign	0.149	Likely Benign	Likely Benign	0.201	Likely Benign										-0.48	Neutral	0.245	Benign	0.138	Benign	5.45	Benign	0.07	Tolerated	4.32	4	1	0	-2.5	30.03
c.1402A>G	M468V (3D Viewer)		GAP	Uncertain		1				-9.461	Likely Pathogenic	0.361	Ambiguous	Likely Benign	0.570	Likely Pathogenic	2.69	Destabilizing	0.1	2.20	Destabilizing	2.45	Destabilizing	0.89	Ambiguous	-1.66	Neutral	0.998	Probably Damaging	0.993	Probably Damaging	-1.21	Pathogenic	0.08	Tolerated	3.37	31	1	2	2.3	-32.06
c.1552T>C	Y518H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.797	Likely Pathogenic	0.943	Likely Pathogenic	Ambiguous	0.496	Likely Benign	2.39	Destabilizing	0.4	0.82	Ambiguous	1.61	Ambiguous	1.31	Destabilizing	-4.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.40	Benign	0.08	Tolerated			0	2	-1.9	-26.03
c.1942T>C	F648L	Likely Pathogenic	GAP	Uncertain		1				-9.296	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.468	Likely Benign	2.71	Destabilizing	0.8	2.08	Destabilizing	2.40	Destabilizing	1.04	Destabilizing	-5.98	Deleterious	0.999	Probably Damaging	0.976	Probably Damaging	3.45	Benign	0.08	Tolerated			2	0	1.0	-34.02
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.2219G>A	R740Q	Likely Benign		Uncertain		1	6-33441684-G-A	4	2.48e-6	-5.195	Likely Benign	0.078	Likely Benign	Likely Benign	0.102	Likely Benign										-0.67	Neutral	0.999	Probably Damaging	0.881	Possibly Damaging	2.60	Benign	0.08	Tolerated	4.32	2	1	1	1.0	-28.06
c.1760G>C	R587T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.697	Likely Pathogenic	0.784	Likely Pathogenic	Likely Benign	0.603	Likely Pathogenic	1.14	Ambiguous	0.2	0.74	Ambiguous	0.94	Ambiguous	0.98	Ambiguous	-4.71	Deleterious	0.998	Probably Damaging	0.847	Possibly Damaging	-1.19	Pathogenic	0.08	Tolerated	3.37	35	-1	-1	3.8	-55.08	227.2	87.4	0.0	0.0	0.5	0.1		X						Potentially Pathogenic	The guanidinium group of Arg587, located on an α helix (res. Glu582-Met603), is constantly rotating and breaking/forming multiple hydrogen bonds and/or salt bridges at the surface intersection of α helices in the WT simulations. The positively charged Arg587 side chain can form a salt bridge with either the carboxylate group of Asp583 or Asp586 in the same helix, or with Glu480 on the opposing short helical loop structure (res. Glu480-Leu482).Importantly, the Arg587 side chain also hydrogen bonds with the backbone carbonyl groups of Ala634 and Asn635, as well as the carboxamide group of Asn635 at the end of another α helix (res. Asp616-Phe636). However, in the variant simulations, the neutral hydroxyl group of the Thr587 side chain is unable to form these salt bridges. Due to its smaller size, it also does not form the hydrogen bonds that the Arg587 side chain could. Instead, the hydroxyl group of Thr587 hydrogen bonds with the backbone carbonyl group of Asp583, which could weaken the integrity of the α helix, although this is not observed in the simulations.Overall, the residue swap could weaken the tertiary structure assembly and negatively affect the overall protein folding process.
c.2111G>A	S704N (3D Viewer)	Likely Benign	GAP	Benign/Likely benign		3	6-33441370-G-A	27	1.67e-5	-5.917	Likely Benign	0.421	Ambiguous	Likely Benign	0.058	Likely Benign	0.48	Likely Benign	0.1	-0.12	Likely Benign	0.18	Likely Benign	0.54	Ambiguous	-0.49	Neutral	0.771	Possibly Damaging	0.275	Benign	3.39	Benign	0.08	Tolerated	3.47	10	1	1	-2.7	27.03	233.2	-29.1	-0.1	0.0	-0.1	0.1						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. However, in the variant simulations, the carboxamide side chain of Asn704 achieves more lasting and numerous hydrogen-bonding interactions with the residues at the helix end, such as Glu706, Ala707, and Leu708. Consequently, the residue swap could strengthen the α-helix secondary structure integrity at the helix end, which could have either positive or negative effects on its function.
c.2699C>T	T900M	Likely Benign		Conflicting		2	6-33443251-C-T	14	8.68e-6	-3.852	Likely Benign	0.176	Likely Benign	Likely Benign	0.015	Likely Benign										-0.81	Neutral	0.060	Benign	0.016	Benign	2.79	Benign	0.08	Tolerated	4.32	4	-1	-1	2.6	30.09
c.3238G>T	A1080S	Likely Benign		Uncertain		1	6-33443790-G-T	1	6.26e-7	-3.277	Likely Benign	0.108	Likely Benign	Likely Benign	0.103	Likely Benign										0.01	Neutral	0.702	Possibly Damaging	0.346	Benign	4.16	Benign	0.08	Tolerated	3.77	5	1	1	-2.6	16.00
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.3572G>A	R1191Q	Likely Benign	Coiled-coil	Uncertain		2	6-33444607-G-A	9	5.58e-6	-1.069	Likely Benign	0.943	Likely Pathogenic	Ambiguous	0.343	Likely Benign										-1.41	Neutral	0.998	Probably Damaging	0.992	Probably Damaging	2.68	Benign	0.08	Tolerated	3.82	4	1	1	1.0	-28.06
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.1631G>A	R544Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438874-G-A	1	6.20e-7	-10.281	Likely Pathogenic	0.596	Likely Pathogenic	Likely Benign	0.542	Likely Pathogenic	0.19	Likely Benign	0.2	0.87	Ambiguous	0.53	Ambiguous	1.40	Destabilizing	-2.41	Neutral	1.000	Probably Damaging	0.997	Probably Damaging	-1.40	Pathogenic	0.09	Tolerated	3.37	35	1	1	1.0	-28.06
c.2405G>A	G802D	Likely Benign	SH3-binding motif	Uncertain		1	6-33442957-G-A	1	6.20e-7	-5.083	Likely Benign	0.476	Ambiguous	Likely Benign	0.153	Likely Benign										-0.38	Neutral	0.126	Benign	0.138	Benign	2.72	Benign	0.09	Tolerated	3.77	5	1	-1	-3.1	58.04
c.2714G>A	R905H	Likely Benign		Uncertain		1	6-33443266-G-A	8	4.96e-6	-4.182	Likely Benign	0.457	Ambiguous	Likely Benign	0.192	Likely Benign										-1.11	Neutral	1.000	Probably Damaging	0.991	Probably Damaging	2.59	Benign	0.09	Tolerated	3.77	5	2	0	1.3	-19.05
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.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.3835G>A	A1279T	Likely Benign		Uncertain		2	6-33447883-G-A	2	1.29e-6	-4.871	Likely Benign	0.071	Likely Benign	Likely Benign	0.178	Likely Benign										-0.30	Neutral	0.001	Benign	0.000	Benign	2.71	Benign	0.09	Tolerated	3.77	5	1	0	-2.5	30.03
c.3962C>A	P1321Q	Likely Benign		Benign		1	6-33451836-C-A	1	6.58e-7	-5.594	Likely Benign	0.079	Likely Benign	Likely Benign	0.055	Likely Benign										-0.74	Neutral	0.659	Possibly Damaging	0.034	Benign	4.24	Benign	0.09	Tolerated	3.77	5	0	-1	-1.9	31.01
c.667A>G	T223A (3D Viewer)		PH	Uncertain		1	6-33435518-A-G	3	1.86e-6	-7.076	In-Between	0.316	Likely Benign	Likely Benign	0.574	Likely Pathogenic	0.30	Likely Benign	0.1	0.77	Ambiguous	0.54	Ambiguous	0.74	Ambiguous	-3.36	Deleterious	0.231	Benign	0.058	Benign	5.74	Benign	0.09	Tolerated	3.41	13	1	0	2.5	-30.03	186.4	44.0	0.0	0.0	0.0	0.0	X	X						Uncertain	The introduced residue Ala223 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Unlike the hydroxyl group of the Thr223 side chain in the WT protein, the methyl side chain of Ala223 cannot form hydrogen bonds with nearby residues Thr228 and Lys207. Without these hydrogen-bonding interactions at the β sheet surface, the secondary structure element becomes unstable and partially unfolds in the variant simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.694G>A	A232T (3D Viewer)		PH	Benign		1	6-33435545-G-A	1	6.20e-7	-7.655	In-Between	0.874	Likely Pathogenic	Ambiguous	0.469	Likely Benign	0.47	Likely Benign	0.1	-0.04	Likely Benign	0.22	Likely Benign	0.61	Ambiguous	-1.42	Neutral	0.608	Possibly Damaging	0.240	Benign	5.80	Benign	0.09	Tolerated	3.40	14	1	0	-2.5	30.03	210.8	-42.0	0.5	0.1	0.4	0.5						X		Uncertain	The hydroxyl group of Thr232, located at the end of an anti-parallel β sheet strand (res. Thr228-Ala232), forms hydrogen bonds with nearby residues Glu217, Cys233, and Cys219 in the variant simulations. These hydrogen-bonding interactions at the β sheet surface contribute to the stability of the secondary structure element and prevent it from unfolding. The new hydrogen bond interactions may be more favorable for structural stability than the steric interactions of the methyl side chain of Ala with the side chains of Gln216 and Cys219 in the WT. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.1142G>T	G381V (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438047-G-T	2	1.25e-6	-5.967	Likely Benign	0.146	Likely Benign	Likely Benign	0.618	Likely Pathogenic	7.16	Destabilizing	1.0	4.10	Destabilizing	5.63	Destabilizing	-0.32	Likely Benign	-0.95	Neutral	0.386	Benign	0.157	Benign	1.32	Pathogenic	0.10	Tolerated	4.32	9	-1	-3	4.6	42.08	214.6	-68.8	0.3	0.7	-0.5	0.3								Uncertain	Gly381 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 valine are rarely tolerated. Although no negative structural effects are observed in the variant simulations, Val381 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 well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1172G>T	G391V (3D Viewer)	Likely Benign	C2	Likely Benign		1	6-33438077-G-T	3	1.86e-6	-6.642	Likely Benign	0.133	Likely Benign	Likely Benign	0.595	Likely Pathogenic	4.23	Destabilizing	1.3	4.81	Destabilizing	4.52	Destabilizing	-0.11	Likely Benign	-0.98	Neutral	0.994	Probably Damaging	0.887	Possibly Damaging	1.32	Pathogenic	0.10	Tolerated	3.69	8	-1	-3	4.6	42.08	228.6	-69.0	0.0	0.8	-0.5	0.3								Uncertain	Gly387 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364 and res. Ala399-Ile411). The Ω loop is assumed to directly interact with the membrane, and it is observed to move arbitrarily throughout the WT solvent simulations. This 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 significant roles in protein functions that require flexibility, and thus hydrophobic residues like valine are rarely tolerated. Although no negative structural effects are visualized in the variant’s simulations, Val391 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. Since the effects on the Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
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.1556A>C	E519A (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-8.557	Likely Pathogenic	0.904	Likely Pathogenic	Ambiguous	0.384	Likely Benign	-0.05	Likely Benign	0.0	0.55	Ambiguous	0.25	Likely Benign	0.00	Likely Benign	-5.23	Deleterious	0.999	Probably Damaging	0.998	Probably Damaging	3.33	Benign	0.10	Tolerated	3.37	35	0	-1	5.3	-58.04	162.4	83.5	-0.1	0.1	-0.2	0.0						X		Potentially Benign	Glu519 is located at the beginning of an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the carboxylate side chain of Glu519 does not make any specific interactions. Accordingly, the Ala residue swap does not show any negative structural effects in the variant simulations. However, it should be noted that Glu519 faces the missing part of the N-terminal in the model, and thus its potential role in maintaining the tertiary structure might be de-emphasized in the current model.
c.2339C>G	S780C	Likely Benign		Uncertain		4	6-33442891-C-G	16	9.94e-6	-7.603	In-Between	0.278	Likely Benign	Likely Benign	0.078	Likely Benign										-1.41	Neutral	0.065	Benign	0.043	Benign	2.59	Benign	0.10	Tolerated	3.64	6	-1	0	3.3	16.06
c.2710A>G	M904V	Likely Benign		Likely Benign		2	6-33443262-A-G	77	4.78e-5	-2.907	Likely Benign	0.112	Likely Benign	Likely Benign	0.058	Likely Benign										-0.33	Neutral	0.039	Benign	0.023	Benign	2.80	Benign	0.10	Tolerated	3.77	5	2	1	2.3	-32.06
c.2729G>C	G910A	Likely Benign		Uncertain		1	6-33443281-G-C	1	6.20e-7	-3.587	Likely Benign	0.361	Ambiguous	Likely Benign	0.209	Likely Benign										-1.43	Neutral	0.999	Probably Damaging	0.999	Probably Damaging	2.78	Benign	0.10	Tolerated	3.77	5	1	0	2.2	14.03
c.2840G>C	G947A	Likely Benign		Likely Benign		1	6-33443392-G-C	28	1.73e-5	-6.511	Likely Benign	0.080	Likely Benign	Likely Benign	0.156	Likely Benign										-0.41	Neutral	0.224	Benign	0.131	Benign	4.97	Benign	0.10	Tolerated	4.32	4	1	0	2.2	14.03
c.2873A>C	H958P	Likely Benign		Benign		1	6-33443425-A-C	2	1.24e-6	-8.369	Likely Pathogenic	0.068	Likely Benign	Likely Benign	0.204	Likely Benign										-0.36	Neutral	0.925	Possibly Damaging	0.316	Benign	4.14	Benign	0.10	Tolerated	3.77	5	0	-2	1.6	-40.02
c.2909A>G	E970G	Likely Benign		Benign		1				-0.167	Likely Benign	0.139	Likely Benign	Likely Benign	0.139	Likely Benign										-0.93	Neutral	0.144	Benign	0.058	Benign	4.09	Benign	0.10	Tolerated	4.32	2	0	-2	3.1	-72.06
c.3116T>C	I1039T	Likely Benign		Uncertain		1	6-33443668-T-C	12	7.43e-6	-2.465	Likely Benign	0.645	Likely Pathogenic	Likely Benign	0.193	Likely Benign										0.45	Neutral	0.004	Benign	0.008	Benign	2.75	Benign	0.10	Tolerated	3.77	5	-1	0	-5.2	-12.05
c.3142G>C	G1048R	Likely Benign		Uncertain		1				-4.305	Likely Benign	0.435	Ambiguous	Likely Benign	0.503	Likely Pathogenic										-0.54	Neutral	0.919	Possibly Damaging	0.728	Possibly Damaging	2.54	Benign	0.10	Tolerated	3.77	5	-2	-3	-4.1	99.14
c.3151G>T	G1051C			Likely Pathogenic		1				-9.050	Likely Pathogenic	0.122	Likely Benign	Likely Benign	0.497	Likely Benign										-0.90	Neutral	0.971	Probably Damaging	0.750	Possibly Damaging	-0.74	Pathogenic	0.10	Tolerated	3.77	5	-3	-3	2.9	46.09
c.3154G>A	G1052R			Uncertain		1				-9.050	Likely Pathogenic	0.383	Ambiguous	Likely Benign	0.497	Likely Benign										-0.41	Neutral	0.990	Probably Damaging	0.798	Possibly Damaging	3.90	Benign	0.10	Tolerated	3.77	5	-2	-3	-4.1	99.14
c.3310C>T	P1104S	Likely Benign		Benign		1	6-33443862-C-T	1	6.54e-7	-2.330	Likely Benign	0.073	Likely Benign	Likely Benign	0.088	Likely Benign										-0.30	Neutral	0.770	Possibly Damaging	0.404	Benign	2.77	Benign	0.10	Tolerated	3.77	5	-1	1	0.8	-10.04
c.3323G>T	S1108I			Uncertain		1	6-33443875-G-T			-3.666	Likely Benign	0.292	Likely Benign	Likely Benign	0.145	Likely Benign										-3.73	Deleterious	0.971	Probably Damaging	0.604	Possibly Damaging	2.44	Pathogenic	0.10	Tolerated	3.77	5	-2	-1	5.3	26.08
c.3355G>A	G1119R			Benign		1	6-33443907-G-A	64	4.23e-5	-8.489	Likely Pathogenic	0.473	Ambiguous	Likely Benign	0.303	Likely Benign										0.10	Neutral	0.969	Probably Damaging	0.462	Possibly Damaging	4.03	Benign	0.10	Tolerated	4.32	2	-3	-2	-4.1	99.14
c.3404A>C	K1135T	Likely Benign		Conflicting		2	6-33443956-A-C	1	6.75e-7	-4.778	Likely Benign	0.779	Likely Pathogenic	Likely Benign	0.210	Likely Benign										-0.90	Neutral	0.411	Benign	0.321	Benign	5.46	Benign	0.10	Tolerated	4.32	2	0	-1	3.2	-27.07
c.3607C>T	H1203Y	Likely Benign	Coiled-coil	Uncertain		1	6-33446599-C-T	2	1.24e-6	-6.834	Likely Benign	0.149	Likely Benign	Likely Benign	0.233	Likely Benign										-1.52	Neutral	0.006	Benign	0.011	Benign	5.55	Benign	0.10	Tolerated	3.77	5	2	0	1.9	26.03
c.3902C>G	P1301R	Likely Benign		Uncertain		1	6-33451776-C-G	15	9.30e-6	-4.753	Likely Benign	0.162	Likely Benign	Likely Benign	0.076	Likely Benign										-1.13	Neutral	0.077	Benign	0.059	Benign	2.81	Benign	0.10	Tolerated	3.77	5	0	-2	-2.9	59.07
c.3980C>T	P1327L	Likely Benign		Uncertain		1	6-33451854-C-T	2	1.28e-6	-5.264	Likely Benign	0.242	Likely Benign	Likely Benign	0.142	Likely Benign										-1.24	Neutral	0.994	Probably Damaging	0.908	Possibly Damaging	4.12	Benign	0.10	Tolerated	3.77	5	-3	-3	5.4	16.04
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.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.1742G>A	R581Q (3D Viewer)	Likely Pathogenic	GAP	Benign		1	6-33440794-G-A	8	4.96e-6	-7.584	In-Between	0.673	Likely Pathogenic	Likely Benign	0.481	Likely Benign	1.31	Ambiguous	0.1	-0.42	Likely Benign	0.45	Likely Benign	0.88	Ambiguous	-2.77	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.21	Pathogenic	0.11	Tolerated	3.37	34	1	1	1.0	-28.06	239.6	53.5	-0.2	0.2	-0.4	0.1		X						Potentially Pathogenic	Arg581 is located on a short α-α loop between two α helices (res. Arg563-Glu578 and res. Glu582-Ser604). In the WT simulations, the guanidinium group of Arg581 forms salt bridges with the carboxylate groups of Asp583 within the same helix, as well as with Glu478 and/or Glu480 on a slightly α-helical loop (res. Glu478-Thr488) preceding another α helix (res. Ala461-Phe476).In the variant simulations, the neutral carboxamide group of the Gln581 side chain cannot form any of these salt bridges. Instead, it packs hydrophobically against Met477 and Ile587 or forms hydrogen bonds sporadically with nearby residues (e.g., Asp583, Arg587). Thus, although no drastic changes are observed in the variant simulations, the residue swap could weaken the tertiary structure assembly.
c.3121C>T	P1041S	Likely Benign		Conflicting		2	6-33443673-C-T	1	6.20e-7	-4.246	Likely Benign	0.121	Likely Benign	Likely Benign	0.344	Likely Benign										-2.72	Deleterious	0.664	Possibly Damaging	0.283	Benign	5.48	Benign	0.11	Tolerated	3.77	5	1	-1	0.8	-10.04
c.3374G>C	G1125A	Likely Benign		Uncertain		1	6-33443926-G-C	1	6.68e-7	-6.569	Likely Benign	0.083	Likely Benign	Likely Benign	0.232	Likely Benign										-0.60	Neutral	0.999	Probably Damaging	0.995	Probably Damaging	4.60	Benign	0.11	Tolerated	3.77	5	1	0	2.2	14.03
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.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.484C>T	R162C			Pathogenic		2				-8.157	Likely Pathogenic	0.787	Likely Pathogenic	Ambiguous	0.150	Likely Benign										-2.05	Neutral	0.988	Probably Damaging	0.513	Possibly Damaging	4.00	Benign	0.11	Tolerated	3.74	4	-4	-3	7.0	-53.05
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.1718G>A	R573Q (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-9.900	Likely Pathogenic	0.923	Likely Pathogenic	Ambiguous	0.733	Likely Pathogenic	2.28	Destabilizing	0.8	1.94	Ambiguous	2.11	Destabilizing	1.08	Destabilizing	-3.16	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.31	Pathogenic	0.12	Tolerated	3.37	35	1	1	1.0	-28.06	230.1	49.9	0.0	0.0	-0.6	0.0	X	X						Potentially Pathogenic	The guanidinium group of Arg573, located in an α-helix (res. Arg563-Glu578), forms a salt bridge with the carboxylate groups of Glu582 and/or Asp586 from a nearby α-helix (res. Glu582-Met603) in the WT simulations. Additionally, the Arg573 side chain stacks planarly with the aromatic phenol ring of Tyr665 and hydrogen bonds with the hydroxyl group of Ser668 from another α-helix (res. Ser641-Ser668). In the variant simulations, although the carboxamide group of the Gln573 side chain can hydrogen bond with the carboxylate group of Glu582 or the hydroxyl group of Ser668, these interactions are not as coordinated, stable, or strong as those of the positively charged Arg573. Consequently, the integrity of the opposing α-helix end (res. Glu582-Met603) is weakened. Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.
c.2195G>C	R732T			Uncertain		1				-8.545	Likely Pathogenic	0.434	Ambiguous	Likely Benign	0.075	Likely Benign										-1.96	Neutral	0.999	Probably Damaging	0.892	Possibly Damaging	2.59	Benign	0.12	Tolerated	3.59	7	-1	-1	3.8	-55.08
c.1752C>G	I584M (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33440804-C-G	1	6.20e-7	-10.119	Likely Pathogenic	0.419	Ambiguous	Likely Benign	0.478	Likely Benign	0.11	Likely Benign	0.1	0.46	Likely Benign	0.29	Likely Benign	1.16	Destabilizing	-2.62	Deleterious	0.983	Probably Damaging	0.925	Probably Damaging	-1.25	Pathogenic	0.12	Tolerated	3.37	34	2	1	-2.6	18.03	247.5	-20.3	-0.1	0.3	-0.1	0.1						X		Potentially Benign	A hydrophobic residue, Ile584, located in an α helix (res. Glu582-Met603), is swapped for another hydrophobic residue, Met584. The sec-butyl hydrocarbon side chain of Ile584 packs hydrophobically with residues in an inter-helix hydrophobic space (e.g., Leu588, Met477, Val473, and Ile483).In the variant simulations, the thioether hydrophobic side chain of Met584 maintains similar interactions as Ile584 in the WT, as it is roughly the same size and fits well within the hydrophobic space. Thus, the residue swap does not appear to cause any negative effects on the protein structure.
c.2014A>G	T672A (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441273-A-G	3	1.86e-6	-6.524	Likely Benign	0.109	Likely Benign	Likely Benign	0.046	Likely Benign	0.51	Ambiguous	0.3	1.15	Ambiguous	0.83	Ambiguous	0.65	Ambiguous	-3.20	Deleterious	0.006	Benign	0.002	Benign	3.44	Benign	0.12	Tolerated	3.40	25	1	0	2.5	-30.03	188.5	42.5	-0.1	0.3	0.2	0.0		X						Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), 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 itself, such as Lys566, Glu666, and Asn669. In the variant simulations, Ala672 can only form a hydrogen bond with Lys566 via its backbone carbonyl group. Consequently, it cannot maintain the Lys566-Glu666 salt bridge through hydrogen bonding, leading to a significant disruption of the intricate and stable hydrogen-bond network between the loop and the helices.
c.2503C>A	L835M	Likely Benign		Benign		1				-4.153	Likely Benign	0.121	Likely Benign	Likely Benign	0.068	Likely Benign										-0.45	Neutral	0.999	Probably Damaging	0.977	Probably Damaging	2.67	Benign	0.12	Tolerated	3.77	5	2	4	-1.9	18.03
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.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.3305C>T	A1102V	Likely Benign		Benign		1	6-33443857-C-T			-2.440	Likely Benign	0.077	Likely Benign	Likely Benign	0.081	Likely Benign										-1.27	Neutral	0.017	Benign	0.028	Benign	2.29	Pathogenic	0.12	Tolerated	3.77	5	0	0	2.4	28.05
c.3379G>A	G1127R	Likely Benign		Uncertain		1	6-33443931-G-A	2	1.34e-6	-5.949	Likely Benign	0.629	Likely Pathogenic	Likely Benign	0.341	Likely Benign										-0.87	Neutral	0.001	Benign	0.001	Benign	4.86	Benign	0.12	Tolerated	4.32	4	-2	-3	-4.1	99.14
c.3379G>C	G1127R	Likely Benign		Conflicting		2	6-33443931-G-C	16	1.07e-5	-5.949	Likely Benign	0.629	Likely Pathogenic	Likely Benign	0.341	Likely Benign										-0.87	Neutral	0.001	Benign	0.001	Benign	4.86	Benign	0.12	Tolerated	4.32	4	-2	-3	-4.1	99.14
c.3662G>A	R1221Q	Likely Benign	Coiled-coil	Conflicting		2	6-33446654-G-A	4	2.48e-6	-5.491	Likely Benign	0.115	Likely Benign	Likely Benign	0.078	Likely Benign										-1.46	Neutral	0.836	Possibly Damaging	0.153	Benign	2.56	Benign	0.12	Tolerated	3.77	5	1	1	1.0	-28.06
c.3686A>C	Q1229P	Likely Pathogenic	Coiled-coil	Uncertain		1				-10.397	Likely Pathogenic	0.980	Likely Pathogenic	Likely Pathogenic	0.422	Likely Benign										-3.69	Deleterious	0.998	Probably Damaging	0.995	Probably Damaging	1.75	Pathogenic	0.12	Tolerated	3.77	5	0	-1	1.9	-31.01
c.485G>A	R162H			Uncertain		1	6-33432782-G-A	2	1.24e-6	-9.730	Likely Pathogenic	0.480	Ambiguous	Likely Benign	0.167	Likely Benign										-1.13	Neutral	0.957	Probably Damaging	0.513	Possibly Damaging	4.03	Benign	0.12	Tolerated	3.74	4	2	0	1.3	-19.05
c.913A>G	T305A (3D Viewer)	Likely Benign	C2	Conflicting		2	6-33437818-A-G	13	8.05e-6	-4.307	Likely Benign	0.078	Likely Benign	Likely Benign	0.144	Likely Benign	1.30	Ambiguous	0.6	1.55	Ambiguous	1.43	Ambiguous	0.77	Ambiguous	-2.10	Neutral	0.939	Possibly Damaging	0.645	Possibly Damaging	1.76	Pathogenic	0.12	Tolerated	3.40	20	1	0	2.5	-30.03	177.9	43.5	-0.2	0.1	0.4	0.0								Uncertain	The hydroxyl group of Thr305, located at the beginning of an anti-parallel β strand (res. Thr305-Asn315), hydrogen bonds with the carboxylate groups of Glu270 and Asp304 in the anti-parallel β strand and the adjacent β hairpin loop, respectively. In the variant simulations, the methyl group of the Ala305 side chain cannot hydrogen bond with either of the acidic residues, which could weaken the integrity of the tertiary structure and the β hairpin loop. Indeed, the guanidinium group of Arg299 does not acquire its central hairpin loop position due to the residue swap.β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant. Due to its location near the membrane surface, the residue swap could also affect the C2 loop dynamics and SynGAP-membrane association. However, this is beyond the scope of the solvent-only simulations to unravel.
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.1789T>C	F597L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.173	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.929	Likely Pathogenic	0.74	Ambiguous	0.1	2.12	Destabilizing	1.43	Ambiguous	1.20	Destabilizing	-5.97	Deleterious	0.999	Probably Damaging	0.994	Probably Damaging	-2.06	Pathogenic	0.13	Tolerated			2	0	1.0	-34.02
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.1625A>G	N542S (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1				-9.675	Likely Pathogenic	0.767	Likely Pathogenic	Likely Benign	0.752	Likely Pathogenic	0.98	Ambiguous	0.1	0.99	Ambiguous	0.99	Ambiguous	0.91	Ambiguous	-4.40	Deleterious	1.000	Probably Damaging	0.989	Probably Damaging	-1.36	Pathogenic	0.13	Tolerated	3.37	35	1	1	2.7	-27.03	212.5	32.1	0.0	0.0	-0.6	0.3		X						Potentially Pathogenic	Asn542 is located in an α-helix (res. Ala533-Val560) next to an α-α loop between two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the carboxamide group of the Asn542 side chain forms a hydrogen bond with the backbone carbonyl group of Asn523 and packs favourably against Glu522 from the loop. In contrast, in the variant simulations, the hydroxyl group of the Ser542 side chain is unable to maintain either the hydrogen bond with Asn523 or the packing against the Glu522 side chain. Instead, the hydroxyl group of Ser542 occasionally forms a hydrogen bond with the backbone carbonyl group of Glu538.Altogether, the residue swap results in a looser helix-loop association, which is especially evident in the third replica simulation, where Asn523 moves away from its initial placement next to the α-helix. In short, based on the simulations, the residue swap weakens the GAP domain tertiary structure assembly, which in turn could negatively affect protein folding.
c.2768T>A	I923N	Likely Benign		Uncertain		1				-0.733	Likely Benign	0.712	Likely Pathogenic	Likely Benign	0.108	Likely Benign										-1.16	Neutral	0.991	Probably Damaging	0.793	Possibly Damaging	2.70	Benign	0.13	Tolerated	3.77	5	-2	-3	-8.0	0.94
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.3304G>C	A1102P	Likely Benign		Uncertain		1				-5.120	Likely Benign	0.077	Likely Benign	Likely Benign	0.118	Likely Benign										-0.97	Neutral	0.000	Benign	0.002	Benign	2.26	Pathogenic	0.13	Tolerated	3.77	5	-1	1	-3.4	26.04
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.1222A>G	T408A		C2	Uncertain		1				-8.304	Likely Pathogenic	0.114	Likely Benign	Likely Benign	0.118	Likely Benign	0.37	Likely Benign	0.6	-0.06	Likely Benign	0.16	Likely Benign	0.72	Ambiguous	-3.07	Deleterious	0.540	Possibly Damaging	0.131	Benign	4.16	Benign	0.14	Tolerated			1	0	2.5	-30.03
c.1312G>A	A438T (3D Viewer)	Likely Benign	GAP	Conflicting		3	6-33438217-G-A	16	9.91e-6	-5.339	Likely Benign	0.085	Likely Benign	Likely Benign	0.021	Likely Benign	0.21	Likely Benign	0.0	-0.07	Likely Benign	0.07	Likely Benign	0.36	Likely Benign	-0.81	Neutral	0.300	Benign	0.011	Benign	4.18	Benign	0.14	Tolerated	3.38	26	1	0	-2.5	30.03	214.2	-42.7	-0.3	0.1	-0.4	0.1						X		Potentially Benign	The methyl group of Ala438, located in a four-residue loop connecting two α helices (res. Asn440-Thr458 and Pro413-Glu436), packs against hydrophobic residues from a nearby α helix or loop residues (e.g., Leu703, Val699). In the variant simulations, the methyl group of Thr438 is able to establish similar hydrophobic packing. Moreover, the hydroxyl group also H-bonds with nearby residues, such as the carbonyl group of the neighboring loop residue Pro437. Accordingly, the residue swap does not generate an apparent negative effect on the protein structure based on the simulations.
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.1678G>A	V560M (3D Viewer)		GAP	Uncertain		2	6-33440730-G-A	15	9.50e-6	-9.598	Likely Pathogenic	0.517	Ambiguous	Likely Benign	0.520	Likely Pathogenic	-0.33	Likely Benign	0.1	0.88	Ambiguous	0.28	Likely Benign	0.72	Ambiguous	-2.42	Neutral	0.999	Probably Damaging	0.863	Possibly Damaging	-1.25	Pathogenic	0.14	Tolerated	3.37	35	2	1	-2.3	32.06	234.9	-52.6	0.0	0.0	-0.1	0.1						X		Potentially Benign	Val560 is located on the surface at the end of an α-helix (res. Ala533-Val560). The iso-propyl group of Val560 favorably packs against Asp508 of the opposing α-helix (res. Gln503-Glu519). However, in the variant simulations, the bulkier thioether side chain of Met560 does not form equally favorable inter-helix interactions. Regardless, no negative structural effects are observed during the simulations.
c.1970G>T	W657L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.411	Likely Pathogenic	0.960	Likely Pathogenic	Likely Pathogenic	0.213	Likely Benign	0.14	Likely Benign	0.1	0.73	Ambiguous	0.44	Likely Benign	0.87	Ambiguous	-10.86	Deleterious	0.277	Benign	0.078	Benign	3.52	Benign	0.14	Tolerated	3.39	24	-2	-2	4.7	-73.05
c.1658A>C	K553T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.328	Likely Pathogenic	0.990	Likely Pathogenic	Likely Pathogenic	0.761	Likely Pathogenic	1.06	Ambiguous	0.2	0.48	Likely Benign	0.77	Ambiguous	0.79	Ambiguous	-5.77	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.34	Pathogenic	0.14	Tolerated	3.37	35	0	-1	3.2	-27.07	218.2	-10.7	0.0	0.0	-0.2	0.5		X						Potentially Pathogenic	Lys533 is located on an α-helix (res. Ala533-Val560). In the WT simulations, Lys533 packs against Phe513, and its amino side chain occasionally forms an ionic interaction with the carboxylate group of Glu512 from an opposing α-helix (res. Gln503-Tyr518). In the variant simulations, Thr533 is unable to reproduce these interactions, potentially weakening the integrity of the tertiary structure. Additionally, Thr533 forms a hydrogen bond with the backbone carbonyl group of Leu549 in the same helix, which could potentially weaken the secondary structure. Regardless, the residue swap does not cause significant structural effects based on the simulations.
c.1667A>G	N556S (3D Viewer)		GAP	Uncertain		1	6-33438910-A-G	3	1.86e-6	-6.576	Likely Benign	0.197	Likely Benign	Likely Benign	0.449	Likely Benign	0.52	Ambiguous	0.1	0.14	Likely Benign	0.33	Likely Benign	0.16	Likely Benign	-3.60	Deleterious	1.000	Probably Damaging	0.989	Probably Damaging	-1.22	Pathogenic	0.14	Tolerated	3.37	35	1	1	2.7	-27.03	198.8	31.0	0.0	0.0	-0.5	0.2						X		Potentially Benign	Asn556 is located on the outer surface of an α-helix (res. Ala533-Val560). The carboxamide group of Asn556 forms hydrogen bonds with nearby residues such as Lys553 and Cys552. It also forms a hydrogen bond with the backbone carbonyl group of Cys552, which weakens the α-helix integrity. In the variant simulations, the hydroxyl group of Ser556 forms a more stable hydrogen bond with the backbone carbonyl oxygen of the same helix residue, Cys552, compared to Asn556 in the WT. Serine has a slightly lower propensity to reside in an α-helix than asparagine, which may exacerbate the negative effect on the α-helix integrity. However, the residue swap does not cause negative structural effects during the 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.1408A>G	M470V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.856	Likely Pathogenic	0.478	Ambiguous	Likely Benign	0.770	Likely Pathogenic	2.73	Destabilizing	0.1	1.88	Ambiguous	2.31	Destabilizing	1.31	Destabilizing	-3.58	Deleterious	0.999	Probably Damaging	0.993	Probably Damaging	-1.20	Pathogenic	0.15	Tolerated	3.37	34	1	2	2.3	-32.06
c.2669G>A	R890H	Likely Benign		Benign		1	6-33443221-G-A	19	1.18e-5	-3.600	Likely Benign	0.198	Likely Benign	Likely Benign	0.056	Likely Benign										-1.29	Neutral	0.254	Benign	0.134	Benign	3.97	Benign	0.15	Tolerated	4.32	4	2	0	1.3	-19.05
c.2818G>C	G940R	Likely Benign		Benign		1	6-33443370-G-C	5	3.10e-6	-6.169	Likely Benign	0.480	Ambiguous	Likely Benign	0.060	Likely Benign										0.02	Neutral	0.922	Possibly Damaging	0.543	Possibly Damaging	2.73	Benign	0.15	Tolerated	3.77	5	-3	-2	-4.1	99.14
c.3731G>A	S1244N	Likely Pathogenic	Coiled-coil	Uncertain		1				-9.008	Likely Pathogenic	0.751	Likely Pathogenic	Likely Benign	0.154	Likely Benign										-1.87	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.10	Pathogenic	0.15	Tolerated	3.77	5	1	1	-2.7	27.03
c.1408A>C	M470L (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438440-A-C	1	6.20e-7	-8.993	Likely Pathogenic	0.406	Ambiguous	Likely Benign	0.678	Likely Pathogenic	0.73	Ambiguous	0.1	0.84	Ambiguous	0.79	Ambiguous	1.04	Destabilizing	-2.72	Deleterious	0.484	Possibly Damaging	0.654	Possibly Damaging	-1.22	Pathogenic	0.16	Tolerated	3.37	34	4	2	1.9	-18.03	225.3	17.9	0.0	0.0	-0.8	0.5						X		Potentially Benign	The thioether group of Met470, located in the middle of an α helix (res. Ala461–Phe476), interacts with hydrophobic residues in the inter-helix space (e.g., Val473, Leu558) formed by two other α helices (res. Ser604–Arg581, res. Pro562–Arg579). In the WT simulations, Met470 also packs against the positively charged guanidinium groups of Arg575, Arg429, and Arg579, which form salt bridges with the negatively charged carboxylate groups of the Asp474 and Asp467 side chains at the protein surface. In the variant simulations, the iso-butyl side chain of Leu470 packs similarly with the hydrophobic residues as methionine, resulting in no negative effects on the protein structure during the simulation.
c.1502T>C	I501T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.996	Likely Benign	0.252	Likely Benign	Likely Benign	0.362	Likely Benign	2.40	Destabilizing	0.1	1.81	Ambiguous	2.11	Destabilizing	1.57	Destabilizing	-3.48	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.44	Benign	0.16	Tolerated	3.37	35	0	-1	-5.2	-12.05	214.5	26.9	0.0	0.0	0.5	0.0							X	Potentially Pathogenic	Ile501 is located near a hinge in the middle of an α-helix (res. Leu489-Glu519). The sec-butyl side chain of Ile501 is hydrophobically packed with other residues in the inter-helix space (e.g., Leu500, Tyr497, Phe679) in the WT simulations. In the variant simulations, the hydroxyl group of Thr501 forms a hydrogen bond with the backbone atoms of Tyr497 on the same α-helix, which may weaken the α-helix integrity. Additionally, the polar hydroxyl group of Thr501 is not suitable for the hydrophobic inter-helix space, and thus, the residue swap could affect protein folding. However, Ile501 is followed by Gly502, which facilitates a hinge in the middle of the α-helix, making further weakening caused by Thr501 unlikely to be harmful to the α-helix integrity.
c.2324G>A	R775Q	Likely Benign		Conflicting		3	6-33442482-G-A	11	1.41e-5	-4.476	Likely Benign	0.229	Likely Benign	Likely Benign	0.085	Likely Benign										-0.63	Neutral	0.969	Probably Damaging	0.863	Possibly Damaging	4.17	Benign	0.16	Tolerated	3.64	6	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
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.3287A>C	E1096A	Likely Benign		Uncertain		1				-4.504	Likely Benign	0.510	Ambiguous	Likely Benign	0.164	Likely Benign										-1.37	Neutral	0.626	Possibly Damaging	0.184	Benign	2.77	Benign	0.16	Tolerated	3.77	5	-1	0	5.3	-58.04
c.3314G>A	R1105Q	Likely Benign		Uncertain		2	6-33443866-G-A	3	1.96e-6	-3.666	Likely Benign	0.216	Likely Benign	Likely Benign	0.104	Likely Benign										-1.21	Neutral	0.958	Probably Damaging	0.194	Benign	2.50	Benign	0.16	Tolerated	3.77	5	1	1	1.0	-28.06
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.970C>T	R324W (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437875-C-T	2	1.24e-6	-12.906	Likely Pathogenic	0.694	Likely Pathogenic	Likely Benign	0.481	Likely Benign	1.49	Ambiguous	0.3	0.56	Ambiguous	1.03	Ambiguous	0.66	Ambiguous	-3.12	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.82	Pathogenic	0.16	Tolerated	3.39	22	2	-3	3.6	30.03	256.6	39.1	0.0	0.1	0.3	0.2		X						Potentially Pathogenic	The guanidinium group of Arg324, located at the end of an anti-parallel β sheet strand (res. Ala322-Asp330), faces outward and frequently forms a salt bridge with the carboxylate group of the Asp288 side chain, which is part of a β strand end (res. Met289-Pro298). In the variant simulations, the indole ring of the Trp324 side chain cannot maintain a similar interaction with the negatively charged carboxylate side chain of Asp288, potentially compromising the folding of the anti-parallel β sheet assembly. However, the residue swap does not appear to negatively impact the protein structure or its integrity based on 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.2702C>T	A901V	Likely Benign		Uncertain		2	6-33443254-C-T	2	1.24e-6	-5.043	Likely Benign	0.219	Likely Benign	Likely Benign	0.029	Likely Benign										-1.83	Neutral	0.106	Benign	0.009	Benign	2.64	Benign	0.17	Tolerated	3.77	5	0	0	2.4	28.05
c.3380G>T	G1127V	Likely Benign		Uncertain		1	6-33443932-G-T	1	6.69e-7	-6.097	Likely Benign	0.094	Likely Benign	Likely Benign	0.230	Likely Benign										-1.01	Neutral	0.004	Benign	0.005	Benign	4.81	Benign	0.17	Tolerated	4.32	4	-1	-3	4.6	42.08
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.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.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