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.382C>A	P128T	Likely Benign		Uncertain		1	6-33432247-C-A	1	6.20e-7	-4.217	Likely Benign	0.267	Likely Benign	Likely Benign	0.075	Likely Benign										-0.96	Neutral	0.952	Possibly Damaging	0.500	Possibly Damaging	4.19	Benign	0.35	Tolerated	3.74	4	-1	0	0.9	3.99
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.1221G>T	Q407H (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-10.526	Likely Pathogenic	0.830	Likely Pathogenic	Ambiguous	0.206	Likely Benign	0.59	Ambiguous	0.0	0.61	Ambiguous	0.60	Ambiguous	1.10	Destabilizing	-4.51	Deleterious	0.982	Probably Damaging	0.947	Probably Damaging	3.88	Benign	0.01	Affected	3.38	28	0	3	0.3	9.01
c.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.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.1153T>C	S385P (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438058-T-C			-5.431	Likely Benign	0.123	Likely Benign	Likely Benign	0.385	Likely Benign	0.91	Ambiguous	0.6	-0.90	Ambiguous	0.01	Likely Benign	0.19	Likely Benign	-0.26	Neutral	0.676	Possibly Damaging	0.693	Possibly Damaging	4.63	Benign	0.04	Affected	4.32	3	1	-1	-0.8	10.04	210.3	18.5	1.8	0.9	0.3	0.0								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycine residues, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Pro385 is potentially tolerated in the Ω loop. However, since the effects on Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1558T>C	S520P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.707	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.855	Likely Pathogenic	3.72	Destabilizing	0.8	8.86	Destabilizing	6.29	Destabilizing	0.83	Ambiguous	-4.57	Deleterious	0.997	Probably Damaging	0.986	Probably Damaging	-1.32	Pathogenic	0.01	Affected			1	-1	-0.8	10.04
c.1600T>C	S534P (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438843-T-C	3	1.86e-6	-5.056	Likely Benign	0.265	Likely Benign	Likely Benign	0.203	Likely Benign	-0.40	Likely Benign	0.2	0.35	Likely Benign	-0.03	Likely Benign	0.47	Likely Benign	-3.81	Deleterious	0.993	Probably Damaging	0.993	Probably Damaging	3.32	Benign	0.05	Affected	3.37	35	-1	1	-0.8	10.04
c.2068T>C	S690P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.568	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.431	Likely Benign	4.84	Destabilizing	0.3	4.40	Destabilizing	4.62	Destabilizing	1.42	Destabilizing	-4.77	Deleterious	0.998	Probably Damaging	0.790	Possibly Damaging	3.44	Benign	0.01	Affected	3.42	17	1	-1	-0.8	10.04	207.5	15.1	0.1	0.0	-0.1	0.2	X	X						Potentially Pathogenic	The hydroxyl side chain of Ser690, located in an α-helix (res. Leu696-Leu685), forms a hydrogen bond with the backbone carbonyl group of Ser410 in an anti-parallel β-sheet of the C2 domain (res. Ile411-Ala399). In the variant simulations, the pyrrolidine side chain of Pro690 cannot form hydrogen bonds with the C2 domain residue, resulting in the loss of this inter-domain connection. Additionally, prolines lack a free amide group necessary for hydrogen bonding with the carbonyl group of Gly686, introducing a slight bend in the α-helix and compromising its integrity.
c.2863T>C	S955P	Likely Benign		Uncertain		1	6-33443415-T-C	3	1.86e-6	-2.584	Likely Benign	0.073	Likely Benign	Likely Benign	0.098	Likely Benign										-0.75	Neutral	0.001	Benign	0.004	Benign	2.33	Pathogenic	0.00	Affected	3.77	5	1	-1	-0.8	10.04
c.313T>C	S105P	Likely Benign		Uncertain		1				-3.631	Likely Benign	0.166	Likely Benign	Likely Benign	0.204	Likely Benign										0.03	Neutral	0.808	Possibly Damaging	0.212	Benign	4.00	Benign	0.00	Affected	4.32	1	-1	1	-0.8	10.04
c.3394T>C	S1132P	Likely Benign		Conflicting		3	6-33443946-T-C	1	6.74e-7	-1.423	Likely Benign	0.144	Likely Benign	Likely Benign	0.301	Likely Benign										0.38	Neutral	0.003	Benign	0.006	Benign	5.40	Benign	0.28	Tolerated	4.32	4	1	-1	-0.8	10.04
c.3424T>C	S1142P	Likely Benign		Likely Benign		1	6-33444459-T-C	1	6.20e-7	-2.713	Likely Benign	0.222	Likely Benign	Likely Benign	0.107	Likely Benign										-2.19	Neutral	0.918	Possibly Damaging	0.761	Possibly Damaging	2.64	Benign	0.00	Affected	4.32	4	-1	1	-0.8	10.04
c.82T>C	S28P	Likely Benign		Uncertain		1				-3.309	Likely Benign	0.051	Likely Benign	Likely Benign	0.047	Likely Benign										1.37	Neutral	0.000	Benign	0.000	Benign	4.53	Benign	0.00	Affected	4.32	1	1	-1	-0.8	10.04
c.703T>C	S235P (3D Viewer)	Likely Pathogenic	PH	Likely Pathogenic		1				-14.857	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.870	Likely Pathogenic	4.02	Destabilizing	0.1	6.91	Destabilizing	5.47	Destabilizing	1.23	Destabilizing	-4.24	Deleterious	0.917	Possibly Damaging	0.446	Benign	5.47	Benign	0.01	Affected	3.40	14	1	-1	-0.8	10.04	201.5	17.0	0.1	0.0	-0.6	0.0		X						Potentially Pathogenic	In the WT, the hydroxyl group of Ser235, located in a β-α loop between an anti-parallel β sheet strand (res. Gly227-Phe231) and an α helix (residues Ala236-Val250), forms hydrogen bonds with the GAP domain loop residue Glu680 and with the backbone amide groups of Ala237 and Glu238 from the α helix. In the variant simulations, the pyrrolidine ring of Pro235 cannot stabilize the α helix end or maintain tertiary bonding interactions between the PH and GAP domains via hydrogen bonding as effectively as serine.
c.2168C>T	T723I (3D Viewer)	Likely Benign	GAP	Likely Benign		1	6-33441633-C-T	2	1.24e-6	-2.591	Likely Benign	0.120	Likely Benign	Likely Benign	0.045	Likely Benign	-0.39	Likely Benign	0.0	-0.20	Likely Benign	-0.30	Likely Benign	0.26	Likely Benign	-2.09	Neutral	0.088	Benign	0.030	Benign	3.39	Benign	0.03	Affected	3.50	8	0	-1	5.2	12.05	252.3	-31.6	0.0	0.0	-0.2	0.2						X		Uncertain	The hydroxyl group of Thr723, located on the outer surface of an α-helix (res. Leu714-Arg726), continuously forms hydrogen bonds with the backbone carbonyl of Asn719 in the WT simulations, potentially lowering the stability of the α-helix. In the variant simulations, the sec-butyl side chain of Ile723 cannot form any hydrogen bonds, which, in theory, could increase the helix stability. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2924C>T	T975I	Likely Benign		Uncertain		1	6-33443476-C-T	6	3.72e-6	-3.912	Likely Benign	0.164	Likely Benign	Likely Benign	0.068	Likely Benign										-1.66	Neutral	0.411	Benign	0.239	Benign	4.11	Benign	0.66	Tolerated	4.32	2	0	-1	5.2	12.05
c.3053C>T	T1018I			Uncertain		1	6-33443605-C-T	4	2.48e-6	-3.264	Likely Benign	0.524	Ambiguous	Likely Benign	0.076	Likely Benign										-2.55	Deleterious	0.586	Possibly Damaging	0.304	Benign	2.24	Pathogenic	0.01	Affected	3.77	5	-1	0	5.2	12.05
c.1040C>A	T347N (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33437945-C-A	9	5.58e-6	-5.545	Likely Benign	0.165	Likely Benign	Likely Benign	0.059	Likely Benign	0.41	Likely Benign	0.1	0.46	Likely Benign	0.44	Likely Benign	-0.06	Likely Benign	1.96	Neutral	0.001	Benign	0.001	Benign	1.67	Pathogenic	0.60	Tolerated	3.37	25	0	0	-2.8	13.00
c.1055C>A	T352N (3D Viewer)	Likely Benign	C2	Likely Benign		1	6-33437960-C-A	2	1.24e-6	-4.817	Likely Benign	0.117	Likely Benign	Likely Benign	0.027	Likely Benign	0.20	Likely Benign	0.0	-0.04	Likely Benign	0.08	Likely Benign	0.45	Likely Benign	-0.92	Neutral	0.255	Benign	0.057	Benign	1.75	Pathogenic	0.19	Tolerated	3.37	25	0	0	-2.8	13.00	208.4	-14.5	-0.2	0.1	-0.1	0.0		X						Potentially Benign	Thr352 is located in a short α helical section within a loop connecting two β strands (res. Gly341-Pro349, res. Thr359-Pro364) originating from two different anti-parallel β sheets of the C2 domain. In the WT simulations, the side chain hydroxyl and backbone amide groups of Thr354 form hydrogen bonds with the backbone carbonyl group of Pro349 at the end of the preceding β strand. This arrangement likely stabilizes the α helical section and aids in folding, keeping the short secondary structure element intact in the variant simulations. However, the carboxamide group of the Asn352 side chain does not form hydrogen bonds with the backbone carbonyl group of Pro349. Instead, it packs against the cyclic ring and forms hydrogen bonds with the phenol group of the Tyr363 side chain in the other β strand.
c.2369C>A	T790N		SH3-binding motif	Conflicting		3	6-33442921-C-A	69	4.28e-5	-5.243	Likely Benign	0.276	Likely Benign	Likely Benign	0.103	Likely Benign										-2.54	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.27	Pathogenic	0.02	Affected	3.64	6	0	0	-2.8	13.00
c.2735C>A	T912N	Likely Benign		Uncertain		1				-4.260	Likely Benign	0.190	Likely Benign	Likely Benign	0.116	Likely Benign										-1.15	Neutral	0.999	Probably Damaging	0.977	Probably Damaging	3.96	Benign	0.00	Affected	3.77	5	0	0	-2.8	13.00
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.458C>A	T153N	Likely Benign		Conflicting		3				-0.739	Likely Benign	0.226	Likely Benign	Likely Benign	0.161	Likely Benign										0.88	Neutral	0.888	Possibly Damaging	0.537	Possibly Damaging	4.23	Benign	0.81	Tolerated	3.61	5	0	0	-2.8	13.00
c.1027G>A	V343I (3D Viewer)	Likely Benign	C2	Uncertain		2	6-33437932-G-A	1	6.20e-7	-6.020	Likely Benign	0.117	Likely Benign	Likely Benign	0.020	Likely Benign	-0.27	Likely Benign	0.0	-0.04	Likely Benign	-0.16	Likely Benign	-0.39	Likely Benign	-0.14	Neutral	0.159	Benign	0.084	Benign	1.98	Pathogenic	0.27	Tolerated	3.37	25	4	3	0.3	14.03	240.2	-26.9	-0.2	0.2	-0.2	0.2						X		Potentially Benign	The iso-propyl side chain of Val343, located in an anti-parallel β sheet strand (res. Gly341-Pro349), is packing against multiple hydrophobic residues of the C2 domain (e.g., Leu327, Leu274, Val365). In the variant simulations, the sec-butyl side chain of Ile343 is basically able to form the same interactions as valine due to its similar hydrophobic profile. The residue swap also does not seem to cause negative effects on the protein structure based on the simulations.
c.103G>A	V35I	Likely Benign		Uncertain		1	6-33423512-G-A	5	3.10e-6	-3.764	Likely Benign	0.081	Likely Benign	Likely Benign	0.017	Likely Benign										-0.32	Neutral	0.672	Possibly Damaging	0.369	Benign	4.16	Benign	0.00	Affected	4.32	1	3	4	0.3	14.03
c.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.1339G>C	V447L (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.136	Likely Benign	0.491	Ambiguous	Likely Benign	0.180	Likely Benign	-1.13	Ambiguous	0.1	0.54	Ambiguous	-0.30	Likely Benign	0.03	Likely Benign	-0.29	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.61	Benign	0.90	Tolerated	3.37	32	1	2	-0.4	14.03
c.1354G>A	V452I (3D Viewer)		GAP	Uncertain		1				-8.985	Likely Pathogenic	0.361	Ambiguous	Likely Benign	0.218	Likely Benign	-0.08	Likely Benign	0.1	0.51	Ambiguous	0.22	Likely Benign	0.25	Likely Benign	-0.99	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.26	Benign	0.05	Affected			4	3	0.3	14.03
c.1417G>A	V473I (3D Viewer)		GAP	Uncertain		1	6-33438449-G-A	1	6.20e-7	-7.481	In-Between	0.418	Ambiguous	Likely Benign	0.203	Likely Benign	-0.12	Likely Benign	0.0	1.20	Ambiguous	0.54	Ambiguous	-0.06	Likely Benign	-0.91	Neutral	0.929	Possibly Damaging	0.917	Probably Damaging	3.74	Benign	0.18	Tolerated	3.37	34	3	4	0.3	14.03
c.1198G>C	V400L (3D Viewer)	Likely Benign	C2	Benign		1	6-33438103-G-C	22	1.36e-5	-1.000	Likely Benign	0.137	Likely Benign	Likely Benign	0.325	Likely Benign	-0.71	Ambiguous	0.2	0.39	Likely Benign	-0.16	Likely Benign	-0.29	Likely Benign	-0.60	Neutral	0.001	Benign	0.001	Benign	5.33	Benign	0.64	Tolerated	3.38	27	2	1	-0.4	14.03	251.0	-30.1	0.0	0.0	0.7	0.1						X		Potentially Benign	The iso-propyl side chain of Val400, located in an anti-parallel β sheet strand (res. Ala399-Ile411), hydrophobically packs against hydrophobic residues within the anti-parallel β sheet of the C2 domain (e.g., Ile268, Ala404, Leu325, Leu402). Val400 is swapped for another hydrophobic residue, leucine, whose branched hydrocarbon side chain is of a comparable size and thus packs favorably within the C2 domain. In short, the residue swap has no apparent negative effect on the structure based on the variant simulations.	10.1016/j.ajhg.2020.11.011
c.1663G>A	V555I (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.544	Likely Benign	0.084	Likely Benign	Likely Benign	0.253	Likely Benign	-0.82	Ambiguous	0.0	-0.41	Likely Benign	-0.62	Ambiguous	-0.55	Ambiguous	0.45	Neutral	0.002	Benign	0.002	Benign	-1.26	Pathogenic	1.00	Tolerated			4	3	0.3	14.03
c.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.1604G>C	S535T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33438847-G-C	14	8.67e-6	-3.886	Likely Benign	0.069	Likely Benign	Likely Benign	0.177	Likely Benign	0.45	Likely Benign	0.1	-0.27	Likely Benign	0.09	Likely Benign	0.17	Likely Benign	-0.81	Neutral	0.000	Benign	0.001	Benign	-1.25	Pathogenic	0.25	Tolerated	3.37	35	1	1	0.1	14.03	201.3	-17.3	-0.1	0.7	-0.2	0.1						X		Potentially Benign	Ser535 is located near the terminal end of an α-helix (res. Ala533-Val560) close to the membrane interface. In the WT simulations, the hydroxyl side chain of Ser535 forms hydrogen bonds with nearby residues (e.g., His539, Glu538) without any specific interactions. These hydrogen bonds disrupt the structure of the terminal end of the α-helix (Ala533-Ser535), causing it to weaken or unfold during the WT simulations. In the variant simulations, Thr535, a hydrophilic residue with a hydroxyl group of almost the same size as Ser, interacts more frequently with the preceding loop residues (e.g., Thr532, Cys531) due to its longer side chain. Regardless, the residue swap is tolerated in the simulations with no negative effects. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.	10.1016/j.ajhg.2020.11.011
c.2111G>C	S704T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.930	Likely Benign	0.265	Likely Benign	Likely Benign	0.071	Likely Benign	0.80	Ambiguous	0.0	0.15	Likely Benign	0.48	Likely Benign	0.29	Likely Benign	-1.72	Neutral	0.525	Possibly Damaging	0.107	Benign	3.45	Benign	0.07	Tolerated	3.47	10	1	1	0.1	14.03	201.7	-18.0	0.0	0.0	-0.2	0.7						X		Potentially Benign	Ser704 is located at the end and outer surface of an α-helix (res. Thr704-Gly712), which is connected via a tight turn or loop to another α-helix (res. Asp684-Gln702). The hydroxyl side chain of Ser704 occasionally forms a hydrogen bond with the amide group of Ala707. Similarly, in the variant simulations, the hydroxyl side chain of Thr704 forms hydrogen bonds with the amide groups of Ala707 and Leu708. Thus, the residue swap does not cause any apparent structural change.
c.2239G>C	V747L	Likely Benign		Uncertain		1	6-33441704-G-C	2	1.24e-6	-2.790	Likely Benign	0.096	Likely Benign	Likely Benign	0.047	Likely Benign										-0.52	Neutral	0.065	Benign	0.033	Benign	2.67	Benign	0.00	Affected	4.32	2	2	1	-0.4	14.03
c.2270G>C	G757A	Likely Benign		Uncertain		1				-2.626	Likely Benign	0.091	Likely Benign	Likely Benign	0.066	Likely Benign										-0.45	Neutral	0.267	Benign	0.127	Benign	2.73	Benign	0.35	Tolerated			1	0	2.2	14.03
c.2362T>A	S788T	Likely Benign	SH3-binding motif	Uncertain		2	6-33442914-T-A	4	2.49e-6	-4.288	Likely Benign	0.288	Likely Benign	Likely Benign	0.092	Likely Benign										-2.25	Neutral	0.979	Probably Damaging	0.982	Probably Damaging	1.55	Pathogenic	0.02	Affected	3.64	6	1	1	0.1	14.03
c.256G>A	V86I	Likely Benign		Uncertain		1				-4.726	Likely Benign	0.338	Likely Benign	Likely Benign	0.076	Likely Benign										-0.31	Neutral	0.267	Benign	0.097	Benign	3.94	Benign	0.00	Affected	4.32	1	4	3	0.3	14.03
c.2578G>A	V860I	Likely Benign		Benign		1	6-33443130-G-A	21	1.30e-5	-4.516	Likely Benign	0.095	Likely Benign	Likely Benign	0.039	Likely Benign										-0.42	Neutral	0.009	Benign	0.006	Benign	4.24	Benign	0.00	Affected	3.77	5	4	3	0.3	14.03
c.2596G>A	V866I	Likely Benign		Conflicting		3	6-33443148-G-A	5	3.10e-6	-4.652	Likely Benign	0.118	Likely Benign	Likely Benign	0.059	Likely Benign										-0.39	Neutral	0.957	Probably Damaging	0.541	Possibly Damaging	2.69	Benign	0.27	Tolerated	3.82	4	4	3	0.3	14.03
c.2596G>T	V866L	Likely Benign		Uncertain		1	6-33443148-G-T	1	6.20e-7	-3.352	Likely Benign	0.148	Likely Benign	Likely Benign	0.046	Likely Benign										-0.97	Neutral	0.217	Benign	0.229	Benign	2.71	Benign	0.21	Tolerated	3.82	4	2	1	-0.4	14.03
c.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.3048C>A	D1016E	Likely Benign		Likely Benign		1	6-33443600-C-A	2	1.24e-6	-3.422	Likely Benign	0.216	Likely Benign	Likely Benign	0.017	Likely Benign										-0.37	Neutral	0.008	Benign	0.028	Benign	2.64	Benign	0.65	Tolerated	3.77	5	2	3	0.0	14.03
c.3176G>C	G1059A	Likely Benign		Uncertain		1	6-33443728-G-C	4	2.49e-6	-6.754	Likely Benign	0.081	Likely Benign	Likely Benign	0.329	Likely Benign										-0.17	Neutral	0.001	Benign	0.002	Benign	2.56	Benign	0.00	Affected	4.32	2	1	0	2.2	14.03
c.335G>C	G112A	Likely Benign		Uncertain		1	6-33432200-G-C	15	9.30e-6	-2.456	Likely Benign	0.119	Likely Benign	Likely Benign	0.114	Likely Benign										-2.34	Neutral	0.231	Benign	0.054	Benign	4.07	Benign	0.00	Affected	3.61	5	1	0	2.2	14.03
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.3380G>C	G1127A	Likely Benign		Conflicting		4	6-33443932-G-C	4	2.68e-6	-5.949	Likely Benign	0.080	Likely Benign	Likely Benign	0.164	Likely Benign										-0.43	Neutral	0.001	Benign	0.002	Benign	4.83	Benign	1.00	Tolerated	4.32	4	1	0	2.2	14.03
c.453C>A	D151E	Likely Benign		Uncertain		1				-5.662	Likely Benign	0.886	Likely Pathogenic	Ambiguous	0.142	Likely Benign										-2.02	Neutral	0.984	Probably Damaging	0.967	Probably Damaging	3.99	Benign	0.11	Tolerated	3.61	5	3	2	0.0	14.03
c.603T>A	D201E (3D Viewer)	Likely Benign	PH	Benign		1				-2.640	Likely Benign	0.406	Ambiguous	Likely Benign	0.165	Likely Benign	0.42	Likely Benign	0.2	1.99	Ambiguous	1.21	Ambiguous	0.23	Likely Benign	-0.69	Neutral	0.633	Possibly Damaging	0.108	Benign	4.30	Benign	1.00	Tolerated	3.46	9	3	2	0.0	14.03	258.7	-24.8	0.9	0.1	-0.3	0.2						X		Uncertain	Asp201, an acidic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another acidic residue, glutamate. The carboxylate groups of both Asp201 and Glu201 side chains form hydrogen bonds with the hydroxyl group of Ser221 in the simulations. Due to its shorter side chain, Asp201 can also hydrogen bond with the backbone amide groups of neighboring loop residues Ser204 and Asp203. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.603T>G	D201E (3D Viewer)	Likely Benign	PH	Conflicting		2	6-33435245-T-G	20	1.24e-5	-2.640	Likely Benign	0.406	Ambiguous	Likely Benign	0.165	Likely Benign	0.42	Likely Benign	0.2	1.99	Ambiguous	1.21	Ambiguous	0.23	Likely Benign	-0.69	Neutral	0.633	Possibly Damaging	0.108	Benign	4.30	Benign	1.00	Tolerated	3.46	9	3	2	0.0	14.03	258.7	-24.8	0.9	0.1	-0.3	0.2						X		Uncertain	Asp201, an acidic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another acidic residue, glutamate. The carboxylate groups of both Asp201 and Glu201 side chains form hydrogen bonds with the hydroxyl group of Ser221 in the simulations. Due to its shorter side chain, Asp201 can also hydrogen bond with the backbone amide groups of neighboring loop residues Ser204 and Asp203. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.70G>A	V24I	Likely Benign		Uncertain		1	6-33423479-G-A	9	5.58e-6	-3.701	Likely Benign	0.137	Likely Benign	Likely Benign	0.069	Likely Benign										-0.25	Neutral	0.043	Benign	0.031	Benign	3.96	Benign	0.00	Affected	4.32	1	3	4	0.3	14.03
c.958G>A	V320I (3D Viewer)	Likely Benign	C2	Uncertain		1				-5.220	Likely Benign	0.111	Likely Benign	Likely Benign	0.027	Likely Benign	-0.27	Likely Benign	0.2	0.66	Ambiguous	0.20	Likely Benign	0.01	Likely Benign	-0.21	Neutral	0.198	Benign	0.114	Benign	1.77	Pathogenic	0.45	Tolerated	3.38	23	3	4	0.3	14.03
c.958G>C	V320L (3D Viewer)		C2	Uncertain		1	6-33437863-G-C	6	3.72e-6	-6.207	Likely Benign	0.362	Ambiguous	Likely Benign	0.096	Likely Benign	-0.26	Likely Benign	0.2	1.33	Ambiguous	0.54	Ambiguous	0.51	Ambiguous	-1.02	Neutral	0.900	Possibly Damaging	0.373	Benign	1.78	Pathogenic	0.92	Tolerated	3.38	23	2	1	-0.4	14.03	245.8	-10.2	0.3	0.9	0.1	0.3						X		Potentially Benign	The isopropyl side chain of Val310, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Thr305-Asn315, res. Ala322-Asp330), hydrophobically packs with the side chains of nearby residues (e.g., Leu286, Val350, Pro318). The hydrophobic Leu320 side chain mostly forms the same interactions; hence, the residue swap does not seem to negatively affect the protein structure based on the variant simulations.
c.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.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.1784T>A	L595Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.101	Likely Pathogenic	0.984	Likely Pathogenic	Likely Pathogenic	0.733	Likely Pathogenic	0.79	Ambiguous	0.1	1.40	Ambiguous	1.10	Ambiguous	1.99	Destabilizing	-5.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.75	Benign	0.00	Affected	3.37	35	-2	-2	-7.3	14.97
c.1964T>A	L655Q (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.278	Likely Benign	0.144	Likely Benign	Likely Benign	0.139	Likely Benign	-0.01	Likely Benign	0.0	0.69	Ambiguous	0.34	Likely Benign	-0.15	Likely Benign	0.61	Neutral	0.955	Possibly Damaging	0.602	Possibly Damaging	3.59	Benign	0.65	Tolerated	3.39	24	-2	-2	-7.3	14.97	229.9	-8.6	0.0	0.0	0.4	0.0						X		Potentially Benign	The iso-butyl side chain of Leu655, located on the surface of an α helix (res. Ser641-Glu666), is not involved in any interactions in the WT simulations. In the variant simulations, the carboxamide side chain of Gln655 dynamically interacts with neighboring residues (e.g., Glu651, Glu656, Arg544) on the protein surface, with no negative structural effects.
c.2075T>A	L692Q (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-13.873	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.596	Likely Pathogenic	3.24	Destabilizing	0.1	3.27	Destabilizing	3.26	Destabilizing	2.76	Destabilizing	-5.98	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.06	Benign	0.00	Affected	3.42	17	-2	-2	-7.3	14.97
c.791T>A	L264Q (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-15.729	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.678	Likely Pathogenic	3.43	Destabilizing	0.1	2.41	Destabilizing	2.92	Destabilizing	2.48	Destabilizing	-5.52	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.49	Pathogenic	0.00	Affected	3.38	18	-2	-2	-7.3	14.97	254.7	-7.6	0.0	0.0	0.0	0.3	X		X				X	Potentially Pathogenic	The iso-butyl branched hydrocarbon side chain of Leu264, located at the end of an anti-parallel β sheet strand (res. Arg259-Arg272), packs against multiple hydrophobic residues such as Leu266, Phe314, Leu317, and Leu323 in the WT simulations. In the variant simulations, the hydrophilic carboxamide group of the Gln264 side chain is not suitable for the hydrophobic niche, causing the hydrophobic residues to make room for the swapped residue. Additionally, the carboxamide group of Gln264 forms hydrogen bonds with the backbone amide groups of Arg405 and Lys256 in the β sheet and the carbonyl group of Val350 in an α helical section of a nearby loop (res. Pro359-Phe358). The residue swap disrupts the packing of the C2 domain, which could adversely affect the C2 domain structure during folding. This disruption could potentially weaken the stability of the SynGAP-membrane association.
c.821T>A	L274Q (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-15.518	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.774	Likely Pathogenic	2.54	Destabilizing	0.3	1.74	Ambiguous	2.14	Destabilizing	1.97	Destabilizing	-5.42	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.00	Pathogenic	0.00	Affected	3.38	19	-2	-2	-7.3	14.97	245.9	1.8	0.0	0.0	0.1	0.2	X		X				X	Potentially Pathogenic	The aliphatic side chain of Leu274, located in a β hairpin loop (res. Glu273-Lys278) connecting two anti-parallel β sheet strands, packs against multiple hydrophobic residues facing the β sheet (e.g., Ala271, Leu327, Tyr280, Val306). The hydrophilic carboxamide group of the Gln274 side chain is not suitable for this hydrophobic niche, causing nearby residues to adjust to make room for the hydrophilic glutamine. Additionally, a new hydrogen bond forms with the backbone carboxyl group of Arg272 in another β strand (res. Glu273-Arg259).As a result, the backbone amide group of Ala399 and the carbonyl group of Arg272, which connect two β strands at the β sheet end, form fewer hydrogen bonds in the variant than in the WT simulations. Although no major secondary structure disruption is observed in the variant simulations, the residue swap could profoundly affect the C2 domain folding, as the hydrophobic packing of Leu274 is crucial for maintaining the loop's contact with the rest of the C2 domain. Lastly, because the Leu274-containing loop faces the membrane surface, the residue swap could also negatively impact the SynGAP-membrane association.
c.3233T>A	V1078D	Likely Benign		Uncertain		1				-5.155	Likely Benign	0.979	Likely Pathogenic	Likely Pathogenic	0.158	Likely Benign										-1.45	Neutral	0.003	Benign	0.008	Benign	3.84	Benign	0.00	Affected	3.77	5	-3	-2	-7.7	15.96
c.917T>A	V306D (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-18.289	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.530	Likely Pathogenic	4.40	Destabilizing	0.3	4.29	Destabilizing	4.35	Destabilizing	2.44	Destabilizing	-5.44	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.74	Pathogenic	0.00	Affected	3.38	19	-2	-3	-7.7	15.96	212.3	-18.3	-0.2	0.4	0.0	0.2	X		X				X	Potentially Pathogenic	The isopropyl group of Val396, located at the beginning of an anti-parallel β sheet strand (res. Thr305-Asn315), packs against multiple hydrophobic residues (e.g., Leu274, Trp308, Ala271) in the WT simulations. However, in the variant simulations, the negatively charged carboxylate group of the Asp306 side chain is not suitable for this hydrophobic niche. Consequently, the side chain moves out to interact with Ser300 in the β strand (res. Met289-Arg299) and the guanidinium group of Arg299 in the β hairpin loop.In the third simulation, the residue swap disrupts the C2 domain secondary structure and tertiary assembly to a large degree when the amino group of the Lys297 side chain rotates to form a salt bridge with Asp306. This drastic effect could potentially reflect the challenge presented by the residue swap during the C2 domain folding. Because the residue swap affects the C2 domain structure, the SynGAP-membrane association could also be impacted. However, this is beyond the scope of the solvent-only simulations to unravel.
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.4021G>T	A1341S	Likely Benign		Uncertain		1	6-33451895-G-T			-2.867	Likely Benign	0.078	Likely Benign	Likely Benign	0.099	Likely Benign										0.80	Neutral	0.000	Benign	0.001	Benign	4.40	Benign	1.00	Tolerated	3.77	5	1	1	-2.6	16.00
c.113C>T	P38L	Likely Benign		Conflicting		4	6-33423522-C-T	8	4.96e-6	-2.469	Likely Benign	0.197	Likely Benign	Likely Benign	0.141	Likely Benign										-2.56	Deleterious	0.983	Probably Damaging	0.931	Probably Damaging	4.02	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
c.1193C>T	P398L (3D Viewer)		C2	Uncertain		1	6-33438098-C-T	8	4.96e-6	-7.518	In-Between	0.547	Ambiguous	Likely Benign	0.599	Likely Pathogenic	1.48	Ambiguous	0.2	-0.54	Ambiguous	0.47	Likely Benign	0.62	Ambiguous	-7.10	Deleterious	0.961	Probably Damaging	0.256	Benign	5.72	Benign	0.01	Affected	3.40	16	-3	-3	5.4	16.04	245.8	-68.6	-0.1	0.0	-0.3	0.2							X	Potentially Pathogenic	Pro398 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. Although the residue swap does not influence the nearby secondary structure elements, proline is often found at the ends of β sheets due to its disfavored status during folding.Additionally, 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 significant roles in protein functions that require flexibility, and thus hydrophobic residues like leucine are rarely tolerated. Although no negative structural effects are visualized in the variant’s simulations, Leu398 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.1685C>T	P562L (3D Viewer)	Likely Pathogenic	GAP	Pathogenic/Likely path.		10	6-33440737-C-T			-13.438	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.829	Likely Pathogenic	3.54	Destabilizing	0.8	0.17	Likely Benign	1.86	Ambiguous	-0.14	Likely Benign	-9.95	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	0.58	Pathogenic	0.00	Affected	3.37	35	-3	-3	5.4	16.04	228.8	-68.5	-0.1	0.0	0.1	0.2							X	Potentially Pathogenic	Pro562 is located on an α-α loop between two α-helices (res. Ala533-Val560 and res. Arg563-Glu578). The cyclic pyrrolidine side chain of Pro562 hydrophobically packs with other residues in the inter-helix space, such as Leu565, Ile501, and Phe561. In the variant simulations, Leu562 packs more favorably with the nearby hydrophobic residues, and the backbone amide group of Leu562 (absent in proline) does not form any intra-protein hydrogen bonds. However, prolines are well-suited for unstructured regions like loops, and thus, Pro562 in the WT is necessary at the end of the helix to induce a tight turn during folding. Although no negative structural effects are observed during the simulations, the residue swap could potentially cause extensive damage to the protein structure during folding.	10.1016/j.ajhg.2020.11.011
c.2381C>T	P794L	Likely Benign	SH3-binding motif	Benign/Likely benign		2	6-33442933-C-T	73	4.52e-5	-3.808	Likely Benign	0.079	Likely Benign	Likely Benign	0.075	Likely Benign										-0.80	Neutral	0.761	Possibly Damaging	0.321	Benign	4.24	Benign	0.03	Affected	4.07	3	-3	-3	5.4	16.04
c.2393C>T	P798L	Likely Benign	SH3-binding motif	Uncertain		2	6-33442945-C-T	6	3.72e-6	-5.640	Likely Benign	0.074	Likely Benign	Likely Benign	0.042	Likely Benign										-0.86	Neutral	0.981	Probably Damaging	0.631	Possibly Damaging	4.21	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
c.266C>T	P89L			Uncertain		2				-6.775	Likely Benign	0.982	Likely Pathogenic	Likely Pathogenic	0.119	Likely Benign										-3.29	Deleterious	0.889	Possibly Damaging	0.058	Benign	3.73	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
c.2822C>T	P941L	Likely Benign		Uncertain		2				-5.692	Likely Benign	0.066	Likely Benign	Likely Benign	0.054	Likely Benign										-0.44	Neutral	0.144	Benign	0.039	Benign	2.76	Benign	0.01	Affected			-3	-3	5.4	16.04
c.2825C>T	P942L	Likely Benign		Uncertain		1	6-33443377-C-T	4	2.48e-6	-5.063	Likely Benign	0.086	Likely Benign	Likely Benign	0.048	Likely Benign										-2.00	Neutral	0.411	Benign	0.239	Benign	2.37	Pathogenic	0.00	Affected	4.32	4	-3	-3	5.4	16.04
c.3194C>T	P1065L			Likely Benign		1	6-33443746-C-T	14	8.71e-6	-5.085	Likely Benign	0.089	Likely Benign	Likely Benign	0.068	Likely Benign										-2.94	Deleterious	0.950	Possibly Damaging	0.419	Benign	2.01	Pathogenic	0.00	Affected	4.32	2	-3	-3	5.4	16.04
c.3197C>T	P1066L	Likely Benign		Likely Benign		1	6-33443749-C-T	14	8.71e-6	-5.478	Likely Benign	0.092	Likely Benign	Likely Benign	0.173	Likely Benign										-3.68	Deleterious	0.996	Probably Damaging	0.903	Possibly Damaging	2.72	Benign	0.00	Affected	4.32	2	-3	-3	5.4	16.04
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.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.3860C>T	P1287L	Likely Benign		Conflicting		2	6-33447908-C-T			-2.800	Likely Benign	0.117	Likely Benign	Likely Benign	0.061	Likely Benign										-1.66	Neutral	0.021	Benign	0.017	Benign	2.76	Benign	0.02	Affected	3.77	5	-3	-3	5.4	16.04
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.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.3974C>T	P1325L	Likely Benign		Uncertain		1	6-33451848-C-T			-5.256	Likely Benign	0.085	Likely Benign	Likely Benign	0.146	Likely Benign										-1.05	Neutral	0.000	Benign	0.000	Benign	4.05	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
c.3977C>T	P1326L	Likely Benign		Uncertain		1				-5.541	Likely Benign	0.115	Likely Benign	Likely Benign	0.117	Likely Benign										-1.06	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	3.62	Benign	0.00	Affected	3.77	5	-3	-3	5.4	16.04
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.59C>T	P20L	Likely Benign		Uncertain		3				-3.289	Likely Benign	0.464	Ambiguous	Likely Benign	0.100	Likely Benign										-0.44	Neutral	0.909	Possibly Damaging	0.713	Possibly Damaging	4.27	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
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.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.227C>G	S76C	Likely Benign		Uncertain		1	6-33425835-C-G	2	1.24e-6	-5.408	Likely Benign	0.100	Likely Benign	Likely Benign	0.076	Likely Benign										-1.78	Neutral	0.992	Probably Damaging	0.869	Possibly Damaging	3.71	Benign	0.00	Affected	4.32	1	0	-1	3.3	16.06
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.2518A>T	S840C	Likely Pathogenic		Uncertain		1				-8.799	Likely Pathogenic	0.904	Likely Pathogenic	Ambiguous	0.376	Likely Benign										-3.96	Deleterious	0.999	Probably Damaging	0.975	Probably Damaging	1.50	Pathogenic	0.00	Affected	3.77	5	0	-1	3.3	16.06
c.2719A>T	S907C	Likely Benign		Likely Benign		1				-6.685	Likely Benign	0.298	Likely Benign	Likely Benign	0.113	Likely Benign										-2.34	Neutral	0.999	Probably Damaging	0.988	Probably Damaging	2.60	Benign	0.02	Affected	3.77	5	0	-1	3.3	16.06
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.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.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.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.1767C>G	I589M (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.225	Likely Pathogenic	0.926	Likely Pathogenic	Ambiguous	0.830	Likely Pathogenic	0.74	Ambiguous	0.2	1.54	Ambiguous	1.14	Ambiguous	1.33	Destabilizing	-2.99	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.94	Pathogenic	0.00	Affected	3.37	35	2	1	-2.6	18.03	267.6	-24.5	0.0	0.0	-0.1	0.1						X		Potentially Benign	A hydrophobic residue, Ile589, located in an α helix (res. Glu582-Met603), is swapped for another hydrophobic residue, methionine. The sec-butyl hydrocarbon side chain of Ile589 packs favourably with multiple residues in the inter-helix hydrophobic space (e.g., Phe569, Ile667, and Leu664).Although the S-methyl thioether group of the Met589 side chain in the variant is longer than the branched side chain of isoleucine, it stacks favourably with the aromatic phenol ring. Additionally, the polar sulphur atom forms a weak hydrogen bond with the guanidinium group of Arg573, which in turn forms a salt bridge with the carboxylate group of Asp586.Overall, the hydrophobic packing in the inter-helix space does not appear to be disrupted in the variant simulations.
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.3721C>A	L1241M		Coiled-coil	Uncertain		1				-5.881	Likely Benign	0.782	Likely Pathogenic	Likely Benign	0.167	Likely Benign										-1.43	Neutral	1.000	Probably Damaging	0.999	Probably Damaging	1.65	Pathogenic	0.00	Affected			4	2	-1.9	18.03
c.1673A>G	H558R (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.445	Likely Pathogenic	0.554	Ambiguous	Likely Benign	0.587	Likely Pathogenic	-1.14	Ambiguous	0.1	-0.23	Likely Benign	-0.69	Ambiguous	1.03	Destabilizing	-4.94	Deleterious	0.677	Possibly Damaging	0.239	Benign	-1.24	Pathogenic	0.14	Tolerated	3.37	35	0	2	-1.3	19.05
c.194A>G	H65R	Likely Benign		Uncertain		1	6-33425802-A-G	1	6.20e-7	-1.980	Likely Benign	0.967	Likely Pathogenic	Likely Pathogenic	0.073	Likely Benign										-1.60	Neutral	0.462	Possibly Damaging	0.227	Benign	4.19	Benign	0.00	Affected	4.32	1	2	0	-1.3	19.05
c.2852A>G	H951R	Likely Benign		Likely Pathogenic		1				-4.964	Likely Benign	0.125	Likely Benign	Likely Benign	0.185	Likely Benign										-1.08	Neutral	0.048	Benign	0.029	Benign	5.46	Benign	0.24	Tolerated	3.77	5	2	0	-1.3	19.05
c.2888A>G	H963R	Likely Benign		Uncertain		1	6-33443440-A-G	8	4.96e-6	-8.952	Likely Pathogenic	0.169	Likely Benign	Likely Benign	0.081	Likely Benign										-1.28	Neutral	0.001	Benign	0.003	Benign	4.15	Benign	0.24	Tolerated	3.77	5	2	0	-1.3	19.05
c.2050G>C	D684H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.194	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.613	Likely Pathogenic	3.36	Destabilizing	1.0	2.95	Destabilizing	3.16	Destabilizing	0.55	Ambiguous	-6.98	Deleterious	1.000	Probably Damaging	0.972	Probably Damaging	3.36	Benign	0.00	Affected	3.42	17	-1	1	0.3	22.05
c.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.451G>C	D151H	Likely Pathogenic		Uncertain		1	6-33432748-G-C	2	1.26e-6	-11.747	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.335	Likely Benign										-3.90	Deleterious	0.999	Probably Damaging	0.995	Probably Damaging	3.86	Benign	0.00	Affected	3.61	5	-1	1	0.3	22.05
c.859G>C	D287H (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-14.518	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.589	Likely Pathogenic	0.48	Likely Benign	0.3	0.32	Likely Benign	0.40	Likely Benign	0.63	Ambiguous	-6.43	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.51	Pathogenic	0.00	Affected	3.38	23	1	-1	0.3	22.05	235.6	3.8	0.1	1.2	0.1	0.1	X	X						Potentially Pathogenic	The carboxylate group of Asp287, located at the beginning of a β hairpin loop connecting two anti-parallel β sheet strands (res. Arg279-Leu286, res. Met289-Pro298), maintains a salt bridge with the guanidinium group of Arg324 in the β sheet during the WT simulations. In the variant simulations, the imidazole ring of the His287 side chain is unable to form a salt bridge with Arg324 or establish any other stable compensatory interactions, which could weaken the beta sandwich assembly of the C2 domain. This destabilization of the C2 domain could adversely affect the stability of the SynGAP-membrane association.
c.1763T>A	L588H (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-16.947	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.939	Likely Pathogenic	4.20	Destabilizing	0.2	3.69	Destabilizing	3.95	Destabilizing	2.26	Destabilizing	-6.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.42	Pathogenic	0.00	Affected	3.38	34	-2	-3	-7.0	23.98	214.3	20.9	0.0	0.0	0.0	0.2	X	X					X	Potentially Pathogenic	The isobutyl group of the Leu588 side chain, located in an α helix (res. Glu582-Met603), packs against hydrophobic residues in the inter-helix hydrophobic space (e.g., Ile584, Trp572, Phe484, Met470, Val473, Ile483).In the variant simulations, the imidazole ring of His588 is aromatic but contains polar delta and epsilon nitrogen atoms that are not suited for the hydrophobic niche. The protonated epsilon nitrogen forms a hydrogen bond with the backbone carbonyl group of Ala469, which can disrupt the continuity of the opposing α helix (res. Phe476-Lys460).While the residue swap could affect the tertiary assembly and the underlying protein folding process, it is difficult to determine if the mutation would be tolerated based solely on the variant simulations.
c.1778T>A	L593H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.504	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.812	Likely Pathogenic	2.52	Destabilizing	0.2	2.32	Destabilizing	2.42	Destabilizing	2.75	Destabilizing	-6.77	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.77	Benign	0.00	Affected	3.37	35	-2	-3	-7.0	23.98	222.0	20.7	0.0	0.0	0.2	0.0		X					X	Potentially Pathogenic	The iso-propyl side chain of Leu593, located in an α helix (res. Glu582-Met603), packs favourably with multiple hydrophobic residues in the inter-helix space (e.g., Leu598, Ile589, Phe594, Phe561).In the variant simulations, His593 retains a similar packing arrangement via its aromatic imidazole ring. However, the polar nitrogen atoms introduce hydrogen bond donors and acceptors into the previously hydrophobic space. The epsilon protonated nitrogen of His593 forms a stable hydrogen bond with the phenol group of the Tyr505 side chain in an α helix (res. Gln503-Tyr518).While the residue swap could affect the tertiary assembly and the underlying protein folding process, it is difficult to determine if the mutation would be tolerated based solely on the variant simulations.
c.1493T>G	M498R (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-8.812	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.869	Likely Pathogenic	3.85	Destabilizing	0.2	2.35	Destabilizing	3.10	Destabilizing	1.76	Destabilizing	-4.53	Deleterious	0.464	Possibly Damaging	0.120	Benign	-1.36	Pathogenic	0.00	Affected			0	-1	-6.4	24.99
c.106C>T	H36Y	Likely Benign		Uncertain		1	6-33423515-C-T	2	1.24e-6	-3.461	Likely Benign	0.139	Likely Benign	Likely Benign	0.023	Likely Benign										-1.03	Neutral	0.219	Benign	0.066	Benign	4.16	Benign	0.00	Affected	4.32	1	0	2	1.9	26.03
c.1441C>T	H481Y (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438473-C-T	16	9.91e-6	-10.910	Likely Pathogenic	0.565	Likely Pathogenic	Likely Benign	0.256	Likely Benign	-0.53	Ambiguous	0.1	-0.46	Likely Benign	-0.50	Ambiguous	0.20	Likely Benign	-3.32	Deleterious	0.988	Probably Damaging	0.979	Probably Damaging	3.40	Benign	0.59	Tolerated	3.37	33	0	2	1.9	26.03	256.5	-44.4	0.0	0.0	0.2	0.2	X			X				Uncertain	The imidazole ring of the His481 side chain is located in a short helical structure (res. Glu480-Leu482) within an α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. In the WT simulations, His481 alternately stacks against Arg485, Arg587, and Glu480 without a definite role. In the variant simulations, Tyr481 also alternately stacks with nearby arginine residues, including Arg485, Arg587, and Arg479. The interaction between Tyr481 and Arg479 affects the α-α loop, causing it to fold into a distorted helical structure, an effect that might be more pronounced during protein folding. Finally, the potential effect of the residue swap on SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.2881C>T	H961Y	Likely Benign		Conflicting		2	6-33443433-C-T	3	1.86e-6	-8.051	Likely Pathogenic	0.157	Likely Benign	Likely Benign	0.102	Likely Benign										-1.07	Neutral	0.716	Possibly Damaging	0.147	Benign	4.10	Benign	0.55	Tolerated	3.77	5	0	2	1.9	26.03
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.502C>T	H168Y	Likely Benign		Benign		1				-8.914	Likely Pathogenic	0.264	Likely Benign	Likely Benign	0.065	Likely Benign										-1.53	Neutral	0.192	Benign	0.062	Benign	4.18	Benign	0.01	Affected	4.32	3	0	2	1.9	26.03
c.88C>T	H30Y	Likely Benign		Uncertain		1				-3.047	Likely Benign	0.115	Likely Benign	Likely Benign	0.082	Likely Benign										-1.84	Neutral	0.273	Benign	0.478	Possibly Damaging	3.99	Benign	0.00	Affected	4.32	1	0	2	1.9	26.03
c.1468G>C	A490P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.905	Likely Pathogenic	0.941	Likely Pathogenic	Ambiguous	0.878	Likely Pathogenic	-1.27	Ambiguous	0.1	1.31	Ambiguous	0.02	Likely Benign	1.07	Destabilizing	-4.81	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.42	Pathogenic	0.01	Affected	3.37	35	-1	1	-3.4	26.04
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.1771G>C	A591P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.479	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.404	Likely Benign	3.78	Destabilizing	0.3	7.29	Destabilizing	5.54	Destabilizing	1.45	Destabilizing	-4.41	Deleterious	0.995	Probably Damaging	0.853	Possibly Damaging	3.35	Benign	0.01	Affected	3.37	35	1	-1	-3.4	26.04	191.5	-10.1	0.2	0.1	0.4	0.1	X							Potentially Pathogenic	The methyl group of the Ala591 side chain, located in the middle of an α helix (res. Glu582-Met603), packs against hydrophobic residues (e.g., Ile483, Phe484) of an opposing partially helical loop (res. Phe476-Asn487).In the variant simulations, Pro591 lacks a free backbone amide group and, therefore, cannot form a hydrogen bond with the backbone carbonyl of Arg587 as Ala591 does in the WT. This notably weakens the α helix integrity and compromises the continuity of the helix. In reality, the effect on the structure during protein folding could be more severe.
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.3964G>C	A1322P	Likely Benign		Benign		1	6-33451838-G-C			-1.153	Likely Benign	0.063	Likely Benign	Likely Benign	0.090	Likely Benign										0.03	Neutral	0.000	Benign	0.000	Benign	4.15	Benign	0.23	Tolerated	3.77	5	1	-1	-3.4	26.04
c.43G>C	A15P	Likely Benign		Uncertain		1				-3.436	Likely Benign	0.097	Likely Benign	Likely Benign	0.146	Likely Benign										-0.23	Neutral	0.880	Possibly Damaging	0.123	Benign	4.09	Benign	0.00	Affected			1	-1	-3.4	26.04
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.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.1154C>T	S385L (3D Viewer)	Likely Benign	C2	Uncertain		2	6-33438059-C-T	9	4.60e-5	-6.018	Likely Benign	0.167	Likely Benign	Likely Benign	0.304	Likely Benign	0.16	Likely Benign	0.1	0.08	Likely Benign	0.12	Likely Benign	-0.26	Likely Benign	-0.68	Neutral	0.829	Possibly Damaging	0.706	Possibly Damaging	4.63	Benign	0.01	Affected	4.32	3	-3	-2	4.6	26.08	244.6	-50.1	0.0	0.6	-0.1	0.1								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like leucine are rarely tolerated. Although no negative structural effects are observed in the variant simulations, Leu385 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effects on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.155C>T	S52L			Uncertain		1	6-33423564-C-T	1	6.20e-7	-7.199	In-Between	0.688	Likely Pathogenic	Likely Benign	0.087	Likely Benign										-1.41	Neutral	0.829	Possibly Damaging	0.706	Possibly Damaging	4.10	Benign	0.00	Affected	4.32	1	-3	-2	4.6	26.08
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.2255C>T	S752L	Likely Benign		Uncertain		2	6-33441720-C-T	6	3.72e-6	-3.386	Likely Benign	0.182	Likely Benign	Likely Benign	0.195	Likely Benign										-2.09	Neutral	0.993	Probably Damaging	0.641	Possibly Damaging	1.51	Pathogenic	0.01	Affected	3.99	5	-3	-2	4.6	26.08
c.1811C>T	S604L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440863-C-T	6	3.72e-6	-14.683	Likely Pathogenic	0.965	Likely Pathogenic	Likely Pathogenic	0.639	Likely Pathogenic	-0.94	Ambiguous	0.1	-1.24	Ambiguous	-1.09	Ambiguous	-0.31	Likely Benign	-5.97	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	3.09	Benign	0.00	Affected	3.37	35	-3	-2	4.6	26.08	234.0	-49.6	0.0	0.1	0.3	0.5	X			X				Potentially Pathogenic	Ser604 is located in a short turn between an α helix (res. Glu582-Met603) and a short α helical section (res. Ser606-Phe608). In the WT simulations, the hydroxyl side chain of Ser604 periodically hydrogen bonds with the backbone carbonyl groups of other α helix residues (e.g., Pro600, Met603). Serine weakens the α helix secondary structure, and thus, Ser604 along with Pro605 breaks the α helix, facilitating the turn in the WT structure.In contrast, in the variant simulations, Leu604 forms a few hydrophobic interactions (e.g., Leu607, Phe608). More importantly, the helix end is more stable than with Ser604 in the WT. The residue swap could have a more profound effect on the actual folding process, for example, by preventing the bending at the α helix end, than what the simulations suggest.Moreover, Ser604 directly hydrogen bonds with Ras residues Ser65 and Ala66 in the WT SynGAP-Ras complex. The hydrophobic leucine cannot maintain these interactions with Ras at the GAP-Ras interface. Thus, the effect of the residue swap on the complex formation with the GTPase cannot be fully explored in the solvent-only simulations.
c.2474C>T	S825L	Likely Pathogenic		Uncertain		1	6-33443026-C-T	1	6.20e-7	-4.987	Likely Benign	0.910	Likely Pathogenic	Ambiguous	0.249	Likely Benign										-4.30	Deleterious	0.999	Probably Damaging	0.994	Probably Damaging	1.94	Pathogenic	0.01	Affected	3.77	5	-2	-3	4.6	26.08
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.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.2690C>T	S897L	Likely Benign		Uncertain		1				-4.034	Likely Benign	0.299	Likely Benign	Likely Benign	0.028	Likely Benign										-1.71	Neutral	0.901	Possibly Damaging	0.636	Possibly Damaging	2.66	Benign	0.01	Affected			-3	-2	4.6	26.08
c.314C>T	S105L	Likely Benign		Uncertain		2	6-33432179-C-T	4	2.48e-6	-3.710	Likely Benign	0.233	Likely Benign	Likely Benign	0.095	Likely Benign										-1.52	Neutral	0.828	Possibly Damaging	0.048	Benign	4.06	Benign	0.00	Affected	4.32	1	-3	-2	4.6	26.08
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.3494C>T	S1165L	Likely Benign		Conflicting		2				-2.984	Likely Benign	0.793	Likely Pathogenic	Ambiguous	0.166	Likely Benign										-2.01	Neutral	0.998	Probably Damaging	0.992	Probably Damaging	2.60	Benign	0.33	Tolerated	3.88	3	-3	-2	4.6	26.08																10.1016/j.ajhg.2020.11.011
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.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.221G>A	S74N	Likely Benign		Uncertain		1	6-33425829-G-A	5	3.10e-6	-5.156	Likely Benign	0.112	Likely Benign	Likely Benign	0.031	Likely Benign										-0.89	Neutral	0.043	Benign	0.007	Benign	4.09	Benign	0.00	Affected	4.32	1	1	1	-2.7	27.03
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.2573G>A	S858N	Likely Benign		Uncertain		1	6-33443125-G-A	2	1.24e-6	-4.311	Likely Benign	0.121	Likely Benign	Likely Benign	0.107	Likely Benign										-0.67	Neutral	0.448	Benign	0.846	Possibly Damaging	4.13	Benign	0.02	Affected	3.77	5	1	1	-2.7	27.03
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.2684G>A	S895N	Likely Benign		Uncertain		1				-6.399	Likely Benign	0.604	Likely Pathogenic	Likely Benign	0.118	Likely Benign										-0.85	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.64	Benign	0.30	Tolerated	4.32	4	1	1	-2.7	27.03
c.2948G>A	S983N			Likely Benign		1	6-33443500-G-A	6	3.72e-6	-5.604	Likely Benign	0.909	Likely Pathogenic	Ambiguous	0.136	Likely Benign										-1.78	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.04	Pathogenic	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.2954G>A	S985N	Likely Benign		Uncertain		1				-6.979	Likely Benign	0.845	Likely Pathogenic	Ambiguous	0.088	Likely Benign										-1.68	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.65	Benign	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.3020G>A	S1007N	Likely Benign		Benign		1				-5.113	Likely Benign	0.803	Likely Pathogenic	Ambiguous	0.075	Likely Benign										-1.54	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.65	Benign	0.01	Affected	3.77	5	1	1	-2.7	27.03
c.3170G>A	S1057N	Likely Benign		Uncertain		1				-6.386	Likely Benign	0.117	Likely Benign	Likely Benign	0.218	Likely Benign										-0.41	Neutral	0.451	Benign	0.129	Benign	5.25	Benign	0.28	Tolerated			1	1	-2.7	27.03
c.3293G>A	S1098N	Likely Benign		Conflicting		2	6-33443845-G-A	6	3.89e-6	-5.120	Likely Benign	0.156	Likely Benign	Likely Benign	0.063	Likely Benign										-0.58	Neutral	0.369	Benign	0.120	Benign	2.76	Benign	0.36	Tolerated	3.77	5	1	1	-2.7	27.03
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.401G>A	S134N	Likely Benign		Uncertain		1				-5.534	Likely Benign	0.813	Likely Pathogenic	Ambiguous	0.075	Likely Benign										-1.62	Neutral	0.001	Benign	0.002	Benign	3.90	Benign	0.00	Affected	3.61	5	1	1	-2.7	27.03
c.416G>A	S139N	Likely Benign		Uncertain		1	6-33432713-G-A	3	2.22e-6	-4.584	Likely Benign	0.688	Likely Pathogenic	Likely Benign	0.109	Likely Benign										-0.75	Neutral	0.149	Benign	0.047	Benign	4.14	Benign	0.24	Tolerated	3.61	5	1	1	-2.7	27.03
c.5G>A	S2N	Likely Benign		Uncertain		2	6-33420269-G-A	3	1.96e-6	-4.104	Likely Benign	0.207	Likely Benign	Likely Benign	0.092	Likely Benign										-0.36	Neutral	0.000	Benign	0.000	Benign	4.06	Benign	0.00	Affected	4.32	1	1	1	-2.7	27.03
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.1511A>G	K504R (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438543-A-G	2	1.24e-6	-4.365	Likely Benign	0.088	Likely Benign	Likely Benign	0.238	Likely Benign	0.13	Likely Benign	0.1	0.51	Ambiguous	0.32	Likely Benign	0.94	Ambiguous	-2.16	Neutral	0.002	Benign	0.015	Benign	-1.41	Pathogenic	0.11	Tolerated	3.37	35	2	3	-0.6	28.01
c.1913A>G	K638R (3D Viewer)	Likely Benign	GAP	Uncertain		1				-2.700	Likely Benign	0.110	Likely Benign	Likely Benign	0.216	Likely Benign	0.09	Likely Benign	0.1	-0.04	Likely Benign	0.03	Likely Benign	0.53	Ambiguous	-2.55	Deleterious	0.649	Possibly Damaging	0.240	Benign	3.41	Benign	0.13	Tolerated	3.37	31	2	3	-0.6	28.01
c.2408A>G	K803R	Likely Benign	SH3-binding motif	Uncertain		1				-2.281	Likely Benign	0.097	Likely Benign	Likely Benign	0.018	Likely Benign										-1.52	Neutral	0.103	Benign	0.038	Benign	2.38	Pathogenic	0.00	Affected	3.77	5	3	2	-0.6	28.01
c.323A>G	K108R	Likely Benign		Uncertain		1	6-33432188-A-G	6	3.72e-6	-2.892	Likely Benign	0.148	Likely Benign	Likely Benign	0.184	Likely Benign										0.37	Neutral	0.993	Probably Damaging	0.956	Probably Damaging	4.22	Benign	1.00	Tolerated	3.61	5	3	2	-0.6	28.01
c.1610C>T	A537V (3D Viewer)	Likely Benign	GAP	Likely Benign		1	6-33438853-C-T	7	4.34e-6	-6.888	Likely Benign	0.120	Likely Benign	Likely Benign	0.382	Likely Benign	0.54	Ambiguous	0.0	-0.05	Likely Benign	0.25	Likely Benign	0.41	Likely Benign	-1.97	Neutral	0.977	Probably Damaging	0.469	Possibly Damaging	-1.26	Pathogenic	0.24	Tolerated	3.37	35	0	0	2.4	28.05	220.3	-45.1	0.0	0.0	-0.7	0.1						X		Potentially Benign	Ala537 is located on the outer surface of an α-helix (res. Ala533-Val560). The methyl group of Ala537 is on the surface and does not form any interactions. In the variant simulations, the iso-propyl side chain of Val537 is also on the surface, similar to Ala537 in the WT, causing no negative structural effects.
c.1802C>T	A601V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.447	Likely Pathogenic	0.853	Likely Pathogenic	Ambiguous	0.535	Likely Pathogenic	1.64	Ambiguous	0.1	0.35	Likely Benign	1.00	Ambiguous	0.81	Ambiguous	-3.98	Deleterious	1.000	Probably Damaging	0.989	Probably Damaging	2.74	Benign	0.03	Affected	3.37	35	0	0	2.4	28.05	228.5	-45.5	0.0	0.0	0.4	0.5						X		Potentially Benign	The methyl side chain of Ala601, located on an α helix (res. Glu582-Met603), packs hydrophobically against other hydrophobic residues in the inter-helix space (e.g., Phe597, Leu598, Leu506, Phe608).In the variant simulations, Val601, which has similar size and physicochemical properties to alanine, resides in the inter-helix hydrophobic space in a similar manner to Ala601 in the WT, causing no apparent negative effect on the protein structure. However, the effect of the residue swap on the SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.2591C>T	A864V	Likely Benign		Uncertain		2	6-33443143-C-T	6	3.72e-6	-4.749	Likely Benign	0.126	Likely Benign	Likely Benign	0.038	Likely Benign										-1.35	Neutral	0.767	Possibly Damaging	0.119	Benign	2.45	Pathogenic	0.30	Tolerated	3.82	4	0	0	2.4	28.05
c.2657C>T	A886V	Likely Benign		Uncertain		1	6-33443209-C-T	18	1.12e-5	-4.478	Likely Benign	0.078	Likely Benign	Likely Benign	0.061	Likely Benign										-0.20	Neutral	0.888	Possibly Damaging	0.314	Benign	2.17	Pathogenic	0.00	Affected	4.32	4	0	0	2.4	28.05
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.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.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.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.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.44C>T	A15V	Likely Benign		Uncertain		1	6-33420308-C-T	1	6.49e-7	-3.560	Likely Benign	0.161	Likely Benign	Likely Benign	0.105	Likely Benign										0.20	Neutral	0.602	Possibly Damaging	0.015	Benign	4.19	Benign	0.00	Affected	4.32	1	0	0	2.4	28.05
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.2105A>G	Q702R (3D Viewer)		GAP	Uncertain		1				-7.894	In-Between	0.348	Ambiguous	Likely Benign	0.294	Likely Benign	-0.31	Likely Benign	0.1	0.63	Ambiguous	0.16	Likely Benign	0.13	Likely Benign	-3.14	Deleterious	0.909	Possibly Damaging	0.889	Possibly Damaging	3.43	Benign	0.02	Affected	3.47	10	1	1	-1.0	28.06	270.3	-52.9	0.0	0.0	0.0	0.1		X						Potentially Pathogenic	The carboxamide side chain of Gln702 is located at the end and outer surface of an α-helix (res. Leu685-Gln702), where it does not directly form hydrogen bonds with any residues in the WT simulations. In the variant simulations, the positively charged guanidinium group of Arg702 forms a salt bridge with the negatively charged carboxylate group of Glu698 on the same helix and/or hydrogen bonds with the backbone carbonyl group of Ala438 on an opposite α-helix (res. Tyr428-Glu436). Consequently, the residue swap could strengthen the tertiary structure assembly, which could have either positive or negative effects on its function.
c.3125A>G	Q1042R	Likely Benign		Uncertain		2	6-33443677-A-G	2	1.24e-6	-2.928	Likely Benign	0.413	Ambiguous	Likely Benign	0.300	Likely Benign										-1.39	Neutral	0.586	Possibly Damaging	0.120	Benign	5.48	Benign	0.12	Tolerated	3.77	5	1	1	-1.0	28.06
c.3773A>G	Q1258R	Likely Pathogenic	Coiled-coil	Uncertain		1				-10.971	Likely Pathogenic	0.931	Likely Pathogenic	Ambiguous	0.316	Likely Benign										-3.19	Deleterious	0.994	Probably Damaging	0.988	Probably Damaging	2.00	Pathogenic	0.00	Affected			1	1	-1.0	28.06
c.1199T>A	V400E (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-13.686	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.810	Likely Pathogenic	3.70	Destabilizing	0.2	2.46	Destabilizing	3.08	Destabilizing	2.29	Destabilizing	-4.88	Deleterious	0.920	Possibly Damaging	0.335	Benign	5.31	Benign	0.00	Affected	3.38	27	-2	-2	-7.7	29.98	249.1	-38.8	-0.1	0.1	1.0	0.0	X		X				X	Potentially Pathogenic	The iso-propyl side chain of Val400, located in an anti-parallel β sheet strand (res. Ala399-Ile411), hydrophobically packs against hydrophobic residues within the anti-parallel β sheet of the C2 domain (e.g., Ile268, Ala404, Leu325, Leu402). In the variant simulations, the negatively charged carboxylate group of the Glu400 side chain is not suitable for occupying the hydrophobic niche. Consequently, the side chain escapes the center of the C2 domain and interacts with the backbone amide groups of Leu402 in the same β strand and/or Ile269 and Glu270 in a neighboring β strand (res. Arg259-Arg272). This residue swap disrupts the hydrophobic packing and generally has extensive negative effects on the C2 domain structure. At a minimum, the residue swap could affect the C2 domain stability and membrane association.
c.269T>A	V90E	Likely Benign		Uncertain		1				-4.079	Likely Benign	0.703	Likely Pathogenic	Likely Benign	0.108	Likely Benign										-0.38	Neutral	0.001	Benign	0.000	Benign	4.00	Benign	0.00	Affected	4.32	1	-2	-2	-7.7	29.98
c.3806T>A	V1269E	Likely Pathogenic	Coiled-coil	Uncertain		1				-11.418	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.403	Likely Benign										-5.05	Deleterious	0.999	Probably Damaging	0.995	Probably Damaging	2.09	Pathogenic	0.00	Affected	3.77	5	-2	-2	-7.7	29.98
c.1030G>A	G344S (3D Viewer)	Likely Pathogenic	C2	Pathogenic		5				-11.254	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.790	Likely Pathogenic	9.02	Destabilizing	0.7	6.08	Destabilizing	7.55	Destabilizing	0.98	Ambiguous	-5.28	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-0.45	Pathogenic	0.04	Affected	3.37	25	1	0	-0.4	30.03	217.3	-51.7	0.0	0.1	0.2	0.1			X				X	Potentially Pathogenic	Because Gly344 lacks a proper side chain, it allows the anti-parallel β sheet strand (res. Gly341-Pro349) to have a slight twist. Within a β strand, side chains normally alternate between outward and inward positions, but glycine is an exception as it allows the alternating pattern to skip a residue. Introducing serine or any other residue with a side chain at position 344 prevents this unique skip in the alternating pattern, causing structural strain or likely preventing correct folding altogether. Additionally, Tyr342 shields Gly344 from the solvent, contributing to twist formation in the β sheet and stabilizing the β-strand.In the variant simulations, the side chain of Ser344 assumes the inward position. However, the hydrophobic niche formed by multiple C2 domain residues (e.g., Val365, Val343, Leu327) is not accommodating for its hydroxyl group. The outward position, not seen in the simulations, would be equally disadvantageous due to the presence of hydrophobic residues on that side as well (e.g., Leu345, Tyr342). Serine is also not well-suited for twist formation, as it tends to suppress twisting and bending in β sheets. At this position, the hydroxyl group of Ser344 could also form hydrogen bonds with the backbone atoms of the Gly-rich Ω loop in the C2 domain (e.g., Thr366, Leu367, Gly378; res. Pro364-Pro398), potentially adversely affecting membrane-loop dynamics and ultimately compromising the stability of the SynGAP-membrane association.
c.1108G>A	G370S (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438013-G-A	15	9.31e-6	-3.533	Likely Benign	0.081	Likely Benign	Likely Benign	0.282	Likely Benign	2.83	Destabilizing	2.0	1.05	Ambiguous	1.94	Ambiguous	-0.02	Likely Benign	0.47	Neutral	0.000	Benign	0.000	Benign	1.33	Pathogenic	0.77	Tolerated	3.42	19	1	0	-0.4	30.03	196.6	-49.6	0.9	2.2	-0.1	0.4								Uncertain	Gly370 is located in the Gly-rich Ω loop (res. Pro364- Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because, the Ω loop is assumed to be directly interacting with the membrane, it is only seen to move arbitrarily throughout the WT solvent simulations. The Ω loop is potentially playing a crucial loop in the SynGAP-membrane complex association, stability and dynamics, regardless, this aspect cannot be addressed through the solvent simulations only. The Ω-loops are known to have a major role in protein functions that requires flexibility and thus, they are rich in glycines, prolines and to a lesser extent, hydrophilic residues to ensure maximum flexibility. Thus, Ser370 in the variant is potentially tolerated in the Ω loop. However, since the effect on the Gly-rich Ω loop dynamics can only be well-studied through the SynGAP-membrane complex, no definite conclusions can be withdrawn.
c.127G>A	G43S	Likely Benign		Uncertain		2	6-33423536-G-A	1	6.20e-7	-3.301	Likely Benign	0.078	Likely Benign	Likely Benign	0.057	Likely Benign										-0.30	Neutral	0.162	Benign	0.096	Benign	4.29	Benign	0.00	Affected	4.32	1	1	0	-0.4	30.03
c.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.1405G>A	A469T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.540	Likely Pathogenic	0.723	Likely Pathogenic	Likely Benign	0.527	Likely Pathogenic	2.26	Destabilizing	0.1	1.90	Ambiguous	2.08	Destabilizing	0.34	Likely Benign	-1.46	Neutral	0.994	Probably Damaging	0.986	Probably Damaging	-1.21	Pathogenic	0.42	Tolerated			1	0	-2.5	30.03
c.1195G>A	A399T (3D Viewer)	Likely Benign	C2	Benign		1				-5.236	Likely Benign	0.114	Likely Benign	Likely Benign	0.272	Likely Benign	1.24	Ambiguous	0.1	0.91	Ambiguous	1.08	Ambiguous	0.49	Likely Benign	-0.40	Neutral	0.131	Benign	0.039	Benign	5.41	Benign	0.69	Tolerated	3.38	26	1	0	-2.5	30.03	211.4	-41.4	0.0	0.0	0.6	0.4		X						Potentially Pathogenic	The methyl group of Ala399, located in an anti-parallel β sheet strand (res. Ala399-Ile411), is swapped for a hydroxyl-containing threonine. In the variant simulations, the hydroxyl group of Thr399 can form H-bonds with the backbone atoms of the residues in the membrane-facing loops (e.g., Gly382) in the C2 domain. Consequently, the ability of the Thr399 side chain to form H-bonds with the membrane-facing loops could adversely affect the dynamics and stability of the SynGAP-membrane association. However, since the effects on the dynamics of the membrane-facing loops can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1285C>T	R429W (3D Viewer)		GAP	Conflicting		5	6-33438190-C-T	65	4.03e-5	-10.666	Likely Pathogenic	0.500	Ambiguous	Likely Benign	0.282	Likely Benign	0.31	Likely Benign	0.1	-0.13	Likely Benign	0.09	Likely Benign	0.52	Ambiguous	-3.19	Deleterious	1.000	Probably Damaging	0.990	Probably Damaging	3.41	Benign	0.03	Affected	3.38	25	2	-3	3.6	30.03	252.3	45.5	0.0	0.0	0.2	0.1		X						Potentially Pathogenic	The guanidinium group of Arg429, located in an α helix (res. Met414-Glu436), either forms a salt bridge with the carboxylate group of an acidic residue (Asp474, Asp467) or a H-bond with the hydroxyl group of Ser471 in an opposing α helix (res. Ala461-Phe476). In the variant simulations, the indole ring of the Trp429 side chain cannot form ionic interactions with the acidic residues. Although it forms a H-bond with Ser471, the bonding is not as strong as that of arginine. The residue swap could affect the tertiary structure assembly during folding; however, no large-scale negative effects were seen during the simulations.
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.1423C>T	R475W (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438455-C-T	1	6.20e-7	-13.235	Likely Pathogenic	0.962	Likely Pathogenic	Likely Pathogenic	0.725	Likely Pathogenic	1.44	Ambiguous	0.4	-0.92	Ambiguous	0.26	Likely Benign	0.56	Ambiguous	-7.56	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.45	Pathogenic	0.00	Affected	3.39	28	2	-3	3.6	30.03	266.9	39.6	0.0	0.0	0.0	0.1	X	X		X				Potentially Pathogenic	In the WT simulations, the guanidinium group of Arg475, located near the end of an α-helix (res. Ala461-Phe476), stacks with the phenyl ring of Phe476 and forms a salt bridge with Glu472. Additionally, Arg475 occasionally forms another salt bridge with the carboxylate group of Glu486 on the α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. Therefore, Arg475 potentially plays a key role in positioning the loop by interacting with Glu486, which is necessary for the positioning of the “arginine finger” (Arg485) and, ultimately, for RasGTPase activation.In the variant simulations, Trp475 moves and stacks with Arg479 on the proceeding α-α loop, disrupting the terminal end of the α-helix. Lastly, the potential effect of the residue swap on the SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
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.1717C>T	R573W (3D Viewer)	Likely Pathogenic	GAP	Conflicting		8				-14.078	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.758	Likely Pathogenic	2.37	Destabilizing	0.7	0.57	Ambiguous	1.47	Ambiguous	0.88	Ambiguous	-6.94	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	-1.48	Pathogenic	0.00	Affected	3.37	35	2	-3	3.6	30.03	257.6	39.0	0.1	0.0	0.2	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, the indole ring of the Trp573 side chain is unable to maintain the same level of coordination as the positively charged Arg573 side chain. Indeed, Trp573 is seen hydrogen bonding only briefly with the carboxylate group of Glu582. 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.1729G>A	A577T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33440781-G-A	6	3.72e-6	-5.311	Likely Benign	0.322	Likely Benign	Likely Benign	0.427	Likely Benign	0.86	Ambiguous	0.1	0.54	Ambiguous	0.70	Ambiguous	0.54	Ambiguous	-1.47	Neutral	0.999	Probably Damaging	0.987	Probably Damaging	-1.31	Pathogenic	0.47	Tolerated	3.37	34	1	0	-2.5	30.03	191.9	-43.4	0.0	0.0	0.7	0.1						X		Potentially Benign	Ala577 is located near the end and outer surface of an α-helix (res. Arg563-Glu578), where its methyl group does not form any particular interactions in the WT simulations. In the variant simulations, the hydroxyl group of the Thr577 side chain hydrogen bonds with the backbone atoms of Arg573 and Lys574 within the same helix, which has the potential to weaken the stability of the secondary structure element. Regardless, the residue swap seems to be well tolerated based on the variant simulations.
c.1741C>T	R581W (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2				-12.855	Likely Pathogenic	0.920	Likely Pathogenic	Ambiguous	0.678	Likely Pathogenic	1.32	Ambiguous	0.1	-0.32	Likely Benign	0.50	Ambiguous	0.68	Ambiguous	-6.79	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	-1.37	Pathogenic	0.01	Affected	3.37	34	2	-3	3.6	30.03	257.8	36.0	0.1	0.1	0.1	0.3	X	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 in a slightly α-helical loop (res. Glu478-Thr488) preceding another α helix (res. Ala461-Phe476).In the variant simulations, the neutral indole ring of the Trp581 side chain cannot form any of these salt bridges. Instead, it packs hydrophobically against Met477 and Ile587 without forming any direct hydrogen bonds. The tendency of the loop (res. Asp477-Thr488) to acquire an α-helical structure seems to marginally increase, potentially due to Trp581's inability to coordinate stable hydrogen bonds with the loop residues (e.g., Glu478-Arg581 salt bridge). Additionally, the residue swap could weaken the tertiary structure assembly and negatively affect the overall protein folding process.
c.2218C>T	R740W			Uncertain		2	6-33441683-C-T	6	3.72e-6	-8.561	Likely Pathogenic	0.168	Likely Benign	Likely Benign	0.180	Likely Benign										-3.09	Deleterious	1.000	Probably Damaging	0.938	Probably Damaging	2.52	Benign	0.01	Affected	4.32	2	2	-3	3.6	30.03
c.2224C>T	R742W	Likely Benign		Uncertain		1	6-33441689-C-T	6	3.72e-6	-7.725	In-Between	0.133	Likely Benign	Likely Benign	0.079	Likely Benign										-1.71	Neutral	0.992	Probably Damaging	0.684	Possibly Damaging	2.66	Benign	0.01	Affected	4.32	2	-3	2	3.6	30.03
c.2245C>T	R749W			Likely Benign		1	6-33441710-C-T	3	1.86e-6	-7.647	In-Between	0.338	Likely Benign	Likely Benign	0.173	Likely Benign										-2.62	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.59	Benign	0.00	Affected	4.32	2	2	-3	3.6	30.03
c.1771G>A	A591T (3D Viewer)	Likely Pathogenic	GAP	Conflicting		3	6-33440823-G-A	18	1.12e-5	-9.572	Likely Pathogenic	0.704	Likely Pathogenic	Likely Benign	0.270	Likely Benign	1.61	Ambiguous	0.2	1.00	Ambiguous	1.31	Ambiguous	1.19	Destabilizing	-3.40	Deleterious	0.955	Possibly Damaging	0.209	Benign	3.48	Benign	0.01	Affected	3.37	35	1	0	-2.5	30.03	202.9	-43.4	0.2	0.0	0.7	0.1						X		Potentially Benign	The methyl group of the Ala591 side chain, located in the middle of an α helix (res. Glu582-Met603), packs against hydrophobic residues (e.g., Ile483, Phe484) of an opposing partially helical loop (res. Phe476-Asn487).In the variant simulations, the hydroxyl group of Thr591 can form hydrogen bonds with the backbone carbonyl of Ile843 in the opposing loop or the backbone carbonyl group of Arg587. These interactions could either reinforce the tertiary assembly or weaken the α helix unity. Additionally, the Thr591 side chain can hydrogen bond with the guanidinium group of the Arg587 side chain, potentially strengthening the α helix unity.Overall, the residue swap does not seem to cause any major negative effects on the protein structure.
c.2350G>A	A784T	Likely Benign		Benign		1				-3.579	Likely Benign	0.089	Likely Benign	Likely Benign	0.046	Likely Benign										1.23	Neutral	0.001	Benign	0.006	Benign	2.92	Benign	1.00	Tolerated	3.64	6	1	0	-2.5	30.03
c.2401G>A	G801S	Likely Benign	SH3-binding motif	Uncertain		1				-3.665	Likely Benign	0.087	Likely Benign	Likely Benign	0.039	Likely Benign										-0.41	Neutral	0.009	Benign	0.019	Benign	2.76	Benign	0.48	Tolerated	4.32	2	0	1	-0.4	30.03
c.2623G>A	A875T	Likely Benign		Uncertain		1	6-33443175-G-A	1	6.20e-7	-3.793	Likely Benign	0.179	Likely Benign	Likely Benign	0.110	Likely Benign										-1.56	Neutral	0.972	Probably Damaging	0.864	Possibly Damaging	2.72	Benign	0.26	Tolerated	3.77	5	0	1	-2.5	30.03
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.2704G>A	A902T	Likely Benign		Likely Benign		1	6-33443256-G-A	36	2.23e-5	-4.966	Likely Benign	0.116	Likely Benign	Likely Benign	0.075	Likely Benign										-1.11	Neutral	0.951	Possibly Damaging	0.617	Possibly Damaging	2.61	Benign	0.01	Affected	3.77	5	1	0	-2.5	30.03
c.2743G>A	G915S	Likely Benign		Benign		1	6-33443295-G-A	9	5.58e-6	-3.557	Likely Benign	0.083	Likely Benign	Likely Benign	0.050	Likely Benign										-0.88	Neutral	0.801	Possibly Damaging	0.201	Benign	2.73	Benign	0.31	Tolerated	3.77	5	1	0	-0.4	30.03
c.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.2818G>A	G940S	Likely Benign		Uncertain		1	6-33443370-G-A	1	6.20e-7	-5.451	Likely Benign	0.084	Likely Benign	Likely Benign	0.135	Likely Benign										0.45	Neutral	0.409	Benign	0.253	Benign	2.77	Benign	0.44	Tolerated	3.77	5	1	0	-0.4	30.03
c.2830G>A	G944S	Likely Benign		Benign		1	6-33443382-G-A	13	8.05e-6	-5.303	Likely Benign	0.082	Likely Benign	Likely Benign	0.223	Likely Benign										-0.75	Neutral	0.007	Benign	0.004	Benign	3.77	Benign	0.00	Affected	4.32	4	1	0	-0.4	30.03
c.2845G>A	G949S	Likely Benign		Benign/Likely benign		4	6-33443397-G-A	122	7.56e-5	-5.693	Likely Benign	0.072	Likely Benign	Likely Benign	0.321	Likely Benign										0.30	Neutral	0.611	Possibly Damaging	0.102	Benign	2.23	Pathogenic	0.00	Affected	4.32	4	1	0	-0.4	30.03																10.1016/j.ajhg.2020.11.011