Data | SynGAP Server

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
c.dna	Variant	SGM Consensus	Domain	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	DOI
c.431C>T	T144M	Likely Pathogenic		Uncertain		2	6-33432728-C-T	2	1.30e-6	-11.228	Likely Pathogenic	0.922	Likely Pathogenic	Ambiguous	0.118	Likely Benign										-3.16	Deleterious	0.913	Possibly Damaging	0.333	Benign	3.73	Benign	0.00	Affected	3.61	5	-1	-1	2.6	30.09													MD Alert	Verdict	Description
c.43G>A	A15T	Likely Benign		Uncertain		1	6-33420307-G-A	4	2.60e-6	-3.720	Likely Benign	0.125	Likely Benign	Likely Benign	0.086	Likely Benign										-0.08	Neutral	0.602	Possibly Damaging	0.017	Benign	4.16	Benign	0.00	Affected	4.32	1	1	0	-2.5	30.03
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.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.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.455G>A	R152Q			Uncertain		1	6-33432752-G-A	5	3.14e-6	-10.336	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.181	Likely Benign										-2.34	Neutral	0.997	Probably Damaging	0.968	Probably Damaging	3.89	Benign	0.00	Affected	3.61	5	1	1	1.0	-28.06
c.467T>G	F156C	Likely Pathogenic		Uncertain		1				-13.658	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.297	Likely Benign										-3.54	Deleterious	0.999	Probably Damaging	0.990	Probably Damaging	3.92	Benign	0.00	Affected			-4	-2	-0.3	-44.04
c.470G>A	R157H			Uncertain		1	6-33432767-G-A	1	6.20e-7	-10.235	Likely Pathogenic	0.604	Likely Pathogenic	Likely Benign	0.254	Likely Benign										-2.23	Neutral	0.999	Probably Damaging	0.987	Probably Damaging	3.80	Benign	0.00	Affected	3.74	4	2	0	1.3	-19.05
c.48G>A	M16I	Likely Benign		Uncertain		1	6-33420312-G-A	1	6.49e-7	-2.198	Likely Benign	0.722	Likely Pathogenic	Likely Benign	0.057	Likely Benign										-0.15	Neutral	0.000	Benign	0.000	Benign	4.28	Benign	0.00	Affected	4.32	1	2	1	2.6	-18.03
c.491G>A	R164Q			Uncertain		1	6-33432788-G-A	2	1.24e-6	-11.208	Likely Pathogenic	0.600	Likely Pathogenic	Likely Benign	0.184	Likely Benign										-1.86	Neutral	0.957	Probably Damaging	0.342	Benign	3.82	Benign	0.00	Affected	3.74	4	1	1	1.0	-28.06
c.508C>T	R170W	Likely Pathogenic		Uncertain		2				-11.660	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.241	Likely Benign										-4.28	Deleterious	0.999	Probably Damaging	0.849	Possibly Damaging	3.84	Benign	0.00	Affected	3.74	4	2	-3	3.6	30.03
c.509G>A	R170Q			Pathogenic/Likely path.		6				-9.021	Likely Pathogenic	0.798	Likely Pathogenic	Ambiguous	0.221	Likely Benign										-2.31	Neutral	0.947	Possibly Damaging	0.342	Benign	3.91	Benign	0.00	Affected	3.74	4	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.50C>T	S17F	Likely Benign		Uncertain		1	6-33420314-C-T	10	6.49e-6	-3.888	Likely Benign	0.637	Likely Pathogenic	Likely Benign	0.048	Likely Benign										-0.99	Neutral	0.486	Possibly Damaging	0.032	Benign	3.99	Benign	0.00	Affected	4.32	1	-2	-3	3.6	60.10
c.514C>T	R172W	Likely Pathogenic		Uncertain		2	6-33435156-C-T	9	5.58e-6	-10.258	Likely Pathogenic	0.878	Likely Pathogenic	Ambiguous	0.228	Likely Benign										-3.61	Deleterious	0.997	Probably Damaging	0.803	Possibly Damaging	3.95	Benign	0.00	Affected	3.61	5	2	-3	3.6	30.03
c.53A>G	Y18C	Likely Benign		Uncertain		1	6-33420317-A-G	44	2.88e-5	-2.658	Likely Benign	0.251	Likely Benign	Likely Benign	0.102	Likely Benign										-0.56	Neutral	0.872	Possibly Damaging	0.206	Benign	4.04	Benign	0.00	Affected	4.32	1	0	-2	3.8	-60.04
c.558G>C	L186F	Likely Pathogenic		Uncertain		1				-11.861	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.132	Likely Benign										-3.03	Deleterious	0.009	Benign	0.012	Benign	3.50	Benign	0.00	Affected			2	0	-1.0	34.02
c.59C>G	P20R	Likely Benign		Uncertain		1				-3.548	Likely Benign	0.434	Ambiguous	Likely Benign	0.146	Likely Benign										-0.15	Neutral	0.972	Probably Damaging	0.804	Possibly Damaging	4.33	Benign	0.00	Affected	4.32	1	0	-2	-2.9	59.07
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.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.662A>G	E221G (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-12.221	Likely Pathogenic	0.992	Likely Pathogenic	Likely Pathogenic	0.863	Likely Pathogenic	1.40	Ambiguous	0.1	1.74	Ambiguous	1.57	Ambiguous	0.71	Ambiguous	-5.56	Deleterious	0.596	Possibly Damaging	0.201	Benign	5.79	Benign	0.00	Affected			0	-2	3.1	-72.06
c.68A>G	D23G	Likely Benign		Uncertain		1				-2.622	Likely Benign	0.684	Likely Pathogenic	Likely Benign	0.100	Likely Benign										-2.45	Neutral	0.805	Possibly Damaging	0.539	Possibly Damaging	3.50	Benign	0.00	Affected			1	-1	3.1	-58.04
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.73C>T	R25W	Likely Benign		Uncertain		2	6-33423482-C-T	6	3.72e-6	-5.133	Likely Benign	0.549	Ambiguous	Likely Benign	0.158	Likely Benign										-1.60	Neutral	0.994	Probably Damaging	0.919	Probably Damaging	3.92	Benign	0.00	Affected	4.32	1	-3	2	3.6	30.03
c.74G>A	R25Q	Likely Benign		Uncertain		1	6-33423483-G-A	15	9.29e-6	-4.126	Likely Benign	0.212	Likely Benign	Likely Benign	0.038	Likely Benign										-0.70	Neutral	0.829	Possibly Damaging	0.614	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06
c.662A>T	E221V (3D Viewer)	Likely Pathogenic	PH	Likely Pathogenic		1				-14.954	Likely Pathogenic	0.987	Likely Pathogenic	Likely Pathogenic	0.875	Likely Pathogenic	-0.66	Ambiguous	0.2	-0.89	Ambiguous	-0.78	Ambiguous	0.49	Likely Benign	-5.54	Deleterious	0.596	Possibly Damaging	0.203	Benign	5.86	Benign	0.00	Affected	3.41	13	-2	-2	7.7	-29.98	234.5	50.6	0.0	0.0	-0.4	0.2		X						Uncertain	The introduced residue Val221 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Unlike the carboxylate group of Glu221, Val221 cannot form hydrogen bonds with Thr223 or a salt bridge with the amino group of the Lys207 side chain. Despite this, the WT simulations containing Glu221 do not show significant differences compared to the variant simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.76G>A	G26R	Likely Benign		Benign		1	6-33423485-G-A	3	1.86e-6	-2.946	Likely Benign	0.678	Likely Pathogenic	Likely Benign	0.189	Likely Benign										-2.22	Neutral	0.994	Probably Damaging	0.990	Probably Damaging	3.87	Benign	0.00	Affected	4.32	1	-3	-2	-4.1	99.14
c.772C>T	R258C (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437677-C-T	1	6.20e-7	-10.285	Likely Pathogenic	0.790	Likely Pathogenic	Ambiguous	0.771	Likely Pathogenic	1.17	Ambiguous	0.4	1.76	Ambiguous	1.47	Ambiguous	0.87	Ambiguous	-6.79	Deleterious	1.000	Probably Damaging	0.993	Probably Damaging	5.77	Benign	0.00	Affected	3.39	15	-3	-4	7.0	-53.05
c.791T>C	L264P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-12.285	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.767	Likely Pathogenic	5.73	Destabilizing	0.3	6.57	Destabilizing	6.15	Destabilizing	2.65	Destabilizing	-6.43	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.49	Pathogenic	0.00	Affected			-3	-3	-5.4	-16.04
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.851T>C	L284P	Likely Pathogenic	C2	Likely Pathogenic		1				-15.588	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.794	Likely Pathogenic	5.83	Destabilizing	0.2	5.81	Destabilizing	5.82	Destabilizing	1.89	Destabilizing	-6.17	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.64	Pathogenic	0.00	Affected			-3	-3	-5.4	-16.04
c.742C>T	R248W (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-11.647	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.699	Likely Pathogenic	1.17	Ambiguous	0.3	-0.20	Likely Benign	0.49	Likely Benign	0.89	Ambiguous	-6.98	Deleterious	1.000	Probably Damaging	0.948	Probably Damaging	5.62	Benign	0.00	Affected	3.41	14	2	-3	3.6	30.03	266.4	42.3	0.0	0.0	0.3	0.1		X						Potentially Pathogenic	The guanidinium group of Arg248, located on an α helix (res. Ala236-Val250), forms two very stable salt bridges with Asp255 (from a short α helical section, res. Lys254-Asn256) and Glu244 (from a nearby loop) in the WT simulations. In the variant simulations, the indole group of Trp248 cannot form any salt bridges, which could negatively affect the tertiary structure assembly of the PH domain. Instead, in the variant simulations, the indole ring of Trp248 stacks against Pro252, which makes a turn after the α helix.
c.743G>C	R248P (3D Viewer)	Likely Pathogenic	PH	Likely Pathogenic		1				-10.751	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.848	Likely Pathogenic	3.09	Destabilizing	0.6	8.87	Destabilizing	5.98	Destabilizing	1.21	Destabilizing	-5.97	Deleterious	0.998	Probably Damaging	0.878	Possibly Damaging	5.64	Benign	0.00	Affected	3.41	14	0	-2	2.9	-59.07	223.8	126.6	0.0	0.0	-0.2	0.1	X	X						Potentially Pathogenic	The guanidinium group of Arg248, located on an α helix (residues Ala236-Val250), forms two very stable salt bridges with Asp255 (from a short α helical section, res. Lys254-Asn256) and Glu244 (from a nearby loop) in the WT simulations. In the variant simulations, the pyrrolidine side chain of Pro248 cannot form any salt bridges, which could negatively affect the tertiary structure assembly of the PH domain. Additionally, Pro248 lacks a free amide group needed for hydrogen bonding with the backbone carbonyl group of Asn245, disrupting the continuity of the α helix.
c.86T>C	M29T	Likely Benign		Uncertain		1				-2.167	Likely Benign	0.122	Likely Benign	Likely Benign	0.199	Likely Benign										-0.37	Neutral	0.018	Benign	0.184	Benign	4.33	Benign	0.00	Affected	4.32	1	-1	-1	-2.6	-30.09
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.775C>T	R259W (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-12.186	Likely Pathogenic	0.985	Likely Pathogenic	Likely Pathogenic	0.691	Likely Pathogenic	1.95	Ambiguous	0.8	0.51	Ambiguous	1.23	Ambiguous	0.51	Ambiguous	-7.35	Deleterious	1.000	Probably Damaging	0.993	Probably Damaging	5.76	Benign	0.00	Affected	3.39	15	2	-3	3.6	30.03	254.0	40.0	0.2	0.2	0.2	0.4	X	X					X	Potentially Pathogenic	The guanidinium group of Arg259, located at the beginning of an anti-parallel β sheet strand (res. Arg259-Arg272), forms salt bridges with the carboxylate groups of Asp684 at the end of an α helix (res. Ile683-Gln702, GAP domain) and Asp261 on the same β strand. The Arg259 side chain also frequently forms hydrogen bonds with the backbone carbonyl groups of Ser257, Asn256, and Asp255. In the variant simulations, the indole ring of the Trp259 side chain cannot form salt bridges or maintain hydrogen bonding with the carboxylate group of Asp684 or other nearby residues. Notably, the amino group of the Lys254 side chain maintains a salt bridge with Asp684 and Glu244 throughout the variant simulations, while it forms a cation-π bond with the indole ring of Trp259 in the variant. This salt bridge is not maintained in the WT simulations. Additionally, the partially or loosely α helical conformation of a lysine-containing loop (res. Lys251-Ser257), which extends to a nearby α helix (res. Met414-Asn426), could be stabilized due to the residue swap. Moreover, the bulky size of the Trp259 side chain requires nearby residues to adjust their positioning to accommodate the introduced residue, weakening the tertiary structure assembly between the C2, PH, and GAP domains. The residue swap potentially causes more severe effects during protein folding or for the SynGAP-membrane interaction than the solvent-only simulations imply.
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.929A>G	E310G (3D Viewer)	Likely Pathogenic	C2	Pathogenic		1				-14.132	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.848	Likely Pathogenic	2.38	Destabilizing	0.7	3.56	Destabilizing	2.97	Destabilizing	0.36	Likely Benign	-6.43	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	1.12	Pathogenic	0.00	Affected	3.38	19	-2	0	3.1	-72.06
c.92G>A	R31Q	Likely Benign		Uncertain		1	6-33423501-G-A	7	4.34e-6	-4.434	Likely Benign	0.136	Likely Benign	Likely Benign	0.051	Likely Benign										-0.92	Neutral	0.829	Possibly Damaging	0.614	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06
c.812C>A	A271D (3D Viewer)	Likely Pathogenic	C2	Pathogenic		1				-18.590	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.706	Likely Pathogenic	4.71	Destabilizing	0.4	2.67	Destabilizing	3.69	Destabilizing	1.59	Destabilizing	-5.52	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.62	Pathogenic	0.00	Affected	3.38	19	0	-2	-5.3	44.01	226.2	-63.4	0.0	0.0	0.9	0.1	X		X	X			X	Potentially Pathogenic	The methyl group of Ala271, located near the end of an anti-parallel β sheet strand (res. Arg259-Arg272), packs against multiple hydrophobic residues such as Val400, Val306, and Leu274 in the WT simulations. In the variant simulations, the carboxylate group of Asp271 is not suitable for the hydrophobic niche, causing the hydrophobic residues to make room for the swapped residue. Additionally, the carboxylate group of the Asp271 side chain forms hydrogen bonds with the backbone amide groups of Arg272 and Ala399 in the β sheet, or even forms a salt bridge with the amino group of the Lys394 side chain. This directly affects the integrity of the anti-parallel β sheet at the end. In short, the residue swap disrupts the C2 domain packing during folding, which could 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.835C>T	R279W (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-11.417	Likely Pathogenic	0.942	Likely Pathogenic	Ambiguous	0.485	Likely Benign	2.00	Destabilizing	0.8	1.47	Ambiguous	1.74	Ambiguous	0.80	Ambiguous	-6.29	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.88	Pathogenic	0.00	Affected	3.39	18	2	-3	3.6	30.03	270.0	38.3	0.1	0.0	0.3	0.0								Uncertain	The guanidinium group of Arg279, located at the beginning of an anti-parallel β sheet strand (res. Arg279-Leu286), can form hydrogen bond with the backbone carbonyl groups of nearby loop residues (e.g., Ser296, Ser331, and As332) and form salt bridges with the carboxylate groups of Asp330 and Asp332. In the WT simulations, Arg279 sporadically forms a salt bridge even with the carboxylate group of Glu613, loosely connecting the C2 domain and GAP domain. Meanwhile, the indole ring of the Trp279 side chain is unable to hydrogen bond with the loop residues in the variant simulations. The lack of hydrogen bond or salt bridge formation with the loop residues could be significant, as Arg279 and the loops face the polar head group region of the membrane. Thus, although Trp279 could interact with the membrane surface as a “lipid anchor,” any changes to the wider loop dynamics could still adversely affect the formation of a stable SynGAP-membrane association. However, no definite conclusions on the effect of the residue swap on the SynGAP-membrane association can be drawn from solvent-only simulations.
c.844T>C	C282R (3D Viewer)	Likely Pathogenic	C2	Pathogenic		2				-16.378	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.466	Likely Benign	3.13	Destabilizing	0.6	1.58	Ambiguous	2.36	Destabilizing	1.70	Destabilizing	-11.03	Deleterious	0.999	Probably Damaging	0.998	Probably Damaging	1.63	Pathogenic	0.00	Affected	3.39	18	-4	-3	-7.0	53.05	297.4	-98.2	-0.1	0.1	0.5	0.0	X		X				X	Potentially Pathogenic	The thiol-containing side chain of Cys282, located at the beginning of an anti-parallel β sheet strand (res. Arg279-Leu286), is packed against multiple hydrophobic residues (e.g., Ile268, Leu284, Trp308, Leu327). In the variant simulations, the bulky side chain of Arg282 with its positively charged guanidinium group is not suitable for this hydrophobic niche. Consequently, the hydrophobic residues must either make room to accommodate Arg282 or it must escape the hydrophobic C2 domain core.As a result, new hydrogen bonds are formed with the backbone carbonyl groups of the surrounding β sheet residues Ala399, Leu325, and His326, which decreases the unity of the secondary structure elements. Notably, it is likely that the residue swap causes major problems during the C2 domain folding that are not visible in the variant simulations. In fact, even increased lability in the C2 domain could adversely affect the establishment of a stable SynGAP-membrane association.
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.859G>T	D287Y (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-12.877	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.663	Likely Pathogenic	0.21	Likely Benign	0.2	0.48	Likely Benign	0.35	Likely Benign	0.27	Likely Benign	-8.27	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.51	Pathogenic	0.00	Affected	3.38	23	-4	-3	2.2	48.09	257.8	-44.4	-0.6	1.6	0.2	0.3	X	X						Potentially Pathogenic	The carboxylate group of Asp287, located at the beginning of a β hairpin loop linking 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 phenol group of the Tyr287 side chain is unable to form a salt bridge with the guanidinium group of Arg324, which could weaken the tertiary structure assembly of the C2 domain. However, the phenol group of Tyr287 frequently stacks with the Arg324 guanidinium side chain, which could help maintain the tertiary structure, especially compared to the D287H variant. The destabilization of the C2 domain could adversely affect the stability of the SynGAP-membrane association.
c.877C>T	R293C (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437782-C-T	3	1.86e-6	-12.844	Likely Pathogenic	0.985	Likely Pathogenic	Likely Pathogenic	0.579	Likely Pathogenic	1.38	Ambiguous	0.1	0.62	Ambiguous	1.00	Ambiguous	0.02	Likely Benign	-7.35	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.46	Pathogenic	0.00	Affected	3.38	23	-4	-3	7.0	-53.05	226.0	96.5	0.0	0.0	0.1	0.1		X		X			X	Potentially Pathogenic	The guanidinium group of the Arg293 side chain, located in an anti-parallel β sheet strand (res. Met289-Pro298), packs against the phenol ring of the Tyr281 side chain or forms a salt bridge with the carboxylate group of Glu283 on the outer side of the C2 domain. The positively charged guanidinium side chain of arginine is on the outside surface of the hydrophobic C2 domain, resulting in a twist in the β strand. Although this twist is maintained in the variant simulations, replacing the positively charged residue with a more hydrophobic one, such as cysteine, could remove the twist during protein folding.Because Arg293 is positioned at the C2 and PH domain interface, the residue swap could significantly impact the tertiary structure assembly. Notably, Arg293 is located at the SynGAP-Ras interface, and its role in complex formation cannot be fully understood through solvent-only simulations.
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.922T>C	W308R (3D Viewer)	Likely Pathogenic	C2	Pathogenic		1				-12.264	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.868	Likely Pathogenic	5.40	Destabilizing	0.5	4.27	Destabilizing	4.84	Destabilizing	1.88	Destabilizing	-12.87	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.48	Pathogenic	0.00	Affected	3.38	19	2	-3	-3.6	-30.03	290.4	-26.7	-0.1	0.1	0.0	0.2	X		X				X	Potentially Pathogenic	The indole ring of Trp308, located in an anti-parallel β sheet strand (res. Thr305-Asn315), packs against multiple hydrophobic residues (e.g., Ile268, Val306, Cys282). The indole group of Trp308 also hydrogen bonds with the backbone atoms of the C2 domain residues forming the anti-parallel β sheet (e.g., Tyr280, Thr294). The guanidinium group of Arg308 is comparably sized to the tryptophan it replaced; however, it is also positively charged.In the variant simulations, the charged side chain remains buried deep in the hydrophobic part of the C2 domain, where it forms new hydrogen bonds with the backbone carbonyl atoms of surrounding residues (e.g., Val306, Ile268). However, the residue swap is likely to disrupt the hydrophobic packing during folding. At a minimum, the residue swap could affect the C2 domain stability and membrane association.
c.924G>C	W308C (3D Viewer)	Likely Pathogenic	C2	Pathogenic/Likely path.		2				-12.791	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.738	Likely Pathogenic	5.56	Destabilizing	0.3	4.38	Destabilizing	4.97	Destabilizing	1.26	Destabilizing	-11.95	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.48	Pathogenic	0.00	Affected	3.38	19	-8	-2	3.4	-83.07	230.8	60.5	-0.3	0.1	-0.4	0.4							X	Potentially Pathogenic	The indole ring of Trp308, located in an anti-parallel β sheet strand (res. Thr305-Asn315), packs against multiple hydrophobic residues (e.g., Ile268, Val306, Cys282). The indole group of Trp308 also hydrogen bonds with the backbone atoms of the C2 domain residues forming the anti-parallel β sheet (e.g., Tyr280, Thr294). The introduced Cys308 is smaller than the tryptophan it replaced. The thiol group of the Cys308 side chain is well-suited for the inner hydrophobic part of the C2 domain. Although the negative effects are essentially missing from the simulations, the side chain size difference between the residues is likely to disrupt the hydrophobic packing during folding. At a minimum, the residue swap could affect the C2 domain stability and membrane association.
c.1003C>T	R335C (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437908-C-T	1	6.20e-7	-14.354	Likely Pathogenic	0.938	Likely Pathogenic	Ambiguous	0.277	Likely Benign	0.53	Ambiguous	0.1	0.85	Ambiguous	0.69	Ambiguous	0.46	Likely Benign	-5.69	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.67	Pathogenic	0.01	Affected	3.38	22	-3	-4	7.0	-53.05
c.1067G>A	R356H (3D Viewer)	Likely Pathogenic	C2	Likely Benign		1	6-33437972-G-A	9	5.66e-6	-11.453	Likely Pathogenic	0.614	Likely Pathogenic	Likely Benign	0.314	Likely Benign	0.59	Ambiguous	0.1	-0.27	Likely Benign	0.16	Likely Benign	1.17	Destabilizing	-4.43	Deleterious	0.999	Probably Damaging	0.987	Probably Damaging	1.70	Pathogenic	0.01	Affected	3.39	22	0	2	1.3	-19.05
c.1118G>A	G373E (3D Viewer)		C2	Uncertain		1				-7.281	In-Between	0.569	Likely Pathogenic	Likely Benign	0.420	Likely Benign	4.13	Destabilizing	3.2	0.52	Ambiguous	2.33	Destabilizing	-0.02	Likely Benign	-0.69	Neutral	0.001	Benign	0.000	Benign	3.90	Benign	0.01	Affected			0	-2	-3.1	72.06
c.1126G>T	G376C		C2	Uncertain		1				-7.686	In-Between	0.125	Likely Benign	Likely Benign	0.560	Likely Pathogenic	2.56	Destabilizing	0.5	0.22	Likely Benign	1.39	Ambiguous	0.16	Likely Benign	-1.15	Neutral	1.000	Probably Damaging	1.000	Probably Damaging	1.32	Pathogenic	0.01	Affected			-3	-3	2.9	46.09
c.1157G>A	G386E (3D Viewer)		C2	Uncertain		1	6-33438062-G-A			-9.286	Likely Pathogenic	0.686	Likely Pathogenic	Likely Benign	0.447	Likely Benign	3.69	Destabilizing	2.9	0.79	Ambiguous	2.24	Destabilizing	0.54	Ambiguous	-0.83	Neutral	0.860	Possibly Damaging	0.354	Benign	3.93	Benign	0.01	Affected	4.32	3	-2	0	-3.1	72.06
c.1214G>C	R405P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-14.206	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.572	Likely Pathogenic	3.11	Destabilizing	0.3	5.19	Destabilizing	4.15	Destabilizing	1.26	Destabilizing	-6.32	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.62	Benign	0.01	Affected	3.38	28	-2	0	2.9	-59.07
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.1121C>A	S374Y (3D Viewer)		C2	Uncertain		1				-7.774	In-Between	0.344	Ambiguous	Likely Benign	0.310	Likely Benign	0.71	Ambiguous	1.2	0.66	Ambiguous	0.69	Ambiguous	-0.02	Likely Benign	-1.18	Neutral	0.875	Possibly Damaging	0.271	Benign	5.41	Benign	0.01	Affected	4.32	13	-3	-2	-0.5	76.10	237.3	-76.9	0.5	0.4	0.5	0.3								Uncertain	Ser374 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus, large and relatively hydrophobic residues like tyrosine are rarely tolerated. Additionally, the hydroxyl group of Tyr374 frequently forms various hydrogen bonds with other loop residues in the variant simulations. Although no negative structural effects are observed in the variant simulations, Tyr374 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1136C>G	S379W (3D Viewer)		C2	Uncertain		1	6-33438041-C-G			-8.898	Likely Pathogenic	0.388	Ambiguous	Likely Benign	0.520	Likely Pathogenic	4.32	Destabilizing	3.4	3.56	Destabilizing	3.94	Destabilizing	0.16	Likely Benign	-1.02	Neutral	0.998	Probably Damaging	0.844	Possibly Damaging	3.82	Benign	0.01	Affected	4.32	11	-2	-3	-0.1	99.14	271.3	-75.7	1.4	1.0	0.6	0.5								Uncertain	Ser379 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like tryptophan are rarely tolerated. Although no major negative structural effects are observed in the variant simulations, Trp379 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn
c.1154C>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.1160G>T	G387V (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438065-G-T	22	1.37e-5	-6.199	Likely Benign	0.153	Likely Benign	Likely Benign	0.390	Likely Benign	5.13	Destabilizing	1.8	6.44	Destabilizing	5.79	Destabilizing	-0.33	Likely Benign	-0.54	Neutral	0.069	Benign	0.077	Benign	1.32	Pathogenic	0.01	Affected	4.32	3	-1	-3	4.6	42.08	207.7	-68.4	-0.7	0.8	-0.5	0.1								Uncertain	Gly387 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364 and res. Ala399-Ile411). The Ω loop is assumed to directly interact with the membrane, and it is observed to move arbitrarily throughout the WT solvent simulations. This loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play significant roles in protein functions that require flexibility, and thus hydrophobic residues like valine are rarely tolerated. Although no negative structural effects are visualized in the variant’s simulations, Val387 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.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.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.1474A>G	K492E (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-16.175	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.510	Likely Pathogenic	1.53	Ambiguous	0.1	1.90	Ambiguous	1.72	Ambiguous	1.42	Destabilizing	-3.98	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.99	Benign	0.01	Affected	3.37	35	1	0	0.4	0.94
c.1483G>A	E495K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.478	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.869	Likely Pathogenic	0.15	Likely Benign	0.2	0.66	Ambiguous	0.41	Likely Benign	0.70	Ambiguous	-3.91	Deleterious	0.999	Probably Damaging	0.994	Probably Damaging	-1.29	Pathogenic	0.01	Affected	3.37	35	1	0	-0.4	-0.94
c.1499T>C	L500P (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-15.898	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.894	Likely Pathogenic	5.91	Destabilizing	0.3	8.90	Destabilizing	7.41	Destabilizing	1.92	Destabilizing	-6.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.37	Pathogenic	0.01	Affected	3.37	35	-3	-3	-5.4	-16.04
c.1214G>A	R405H (3D Viewer)	Likely Pathogenic	C2	Conflicting		2	6-33438119-G-A	4	2.48e-6	-9.081	Likely Pathogenic	0.706	Likely Pathogenic	Likely Benign	0.371	Likely Benign	2.79	Destabilizing	0.6	1.85	Ambiguous	2.32	Destabilizing	1.26	Destabilizing	-4.54	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	3.65	Benign	0.01	Affected	3.38	28	2	0	1.3	-19.05	214.0	102.2	-0.1	0.0	-0.7	0.1		X						Potentially Pathogenic	The guanidinium group of Arg405, located in an anti-parallel β sheet strand of the C2 domain (res. Pro398-Ile411), forms a salt bridge with the carboxylate group of the Glu446 side chain from an opposing α helix (res. Val441-Ser457) in the GAP domain. The positively charged Arg405 side chain also stacks with the aromatic ring of the Phe358 side chain from a loop preceding the β strand (res. Thr359-Thr366), which could assist in maintaining the anti-parallel strand arrangement.In the variant simulations, the imidazole ring of His405 does not stack with the aromatic ring of Phe358 nor form any lasting H-bonds with the loop residues. The imidazole ring of His405 (neutral and epsilon protonated in the simulations) is unable to form a salt bridge with Glu446, which could affect the tertiary structure assembly, although this is not apparent based on the variant simulations.
c.1516C>T	L506F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.262	Likely Pathogenic	0.883	Likely Pathogenic	Ambiguous	0.464	Likely Benign	4.92	Destabilizing	0.8	5.76	Destabilizing	5.34	Destabilizing	0.91	Ambiguous	-3.98	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.62	Pathogenic	0.01	Affected	3.37	35	0	2	-1.0	34.02
c.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.1403T>C	M468T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33438435-T-C	1	6.20e-7	-12.399	Likely Pathogenic	0.862	Likely Pathogenic	Ambiguous	0.801	Likely Pathogenic	3.47	Destabilizing	0.1	3.10	Destabilizing	3.29	Destabilizing	1.84	Destabilizing	-3.85	Deleterious	0.994	Probably Damaging	0.985	Probably Damaging	-1.31	Pathogenic	0.01	Affected	3.37	31	-1	-1	-2.6	-30.09	214.6	47.1	0.0	0.0	0.1	0.0							X	Potentially Pathogenic	The thioether group of Met468, located in the middle of an α helix (res. Ala461–Phe476), interacts with hydrophobic residues (e.g., Phe464, Leu465, Leu489) in an inter-helix space formed by two other α helices (res. Ala461–Phe476, res. Thr488–Gly502). In the variant simulations, the hydrophilic side chain of Thr468 does not pack favorably in the hydrophobic niche, and the methionine-aromatic stacking is lost. Although the hydroxyl group of Thr468 forms an H-bond with the backbone carbonyl group of Phe464, the integrity of the α helix is not affected in the simulations. No large-scale structural changes are observed during the variant simulations; however, due to the importance of hydrophobic packing, the effects could be more pronounced during protein folding.
c.1424G>A	R475Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33438456-G-A	5	3.10e-6	-12.087	Likely Pathogenic	0.721	Likely Pathogenic	Likely Benign	0.632	Likely Pathogenic	0.71	Ambiguous	0.1	0.12	Likely Benign	0.42	Likely Benign	0.82	Ambiguous	-3.65	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	-1.32	Pathogenic	0.01	Affected	3.39	28	1	1	1.0	-28.06	253.6	52.7	0.0	0.0	-0.8	0.0	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, Asn475 forms a hydrogen bond with Arg479 on the proceeding α-α loop. The absence of Phe476/Arg475 stacking and the Arg475-Glu472 salt bridge weakens the integrity of the terminal end of the α-helix during the variant simulations. 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.1465C>T	L489F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33438497-C-T	1	6.20e-7	-12.066	Likely Pathogenic	0.965	Likely Pathogenic	Likely Pathogenic	0.724	Likely Pathogenic	1.72	Ambiguous	0.5	1.14	Ambiguous	1.43	Ambiguous	0.56	Ambiguous	-3.76	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	-1.51	Pathogenic	0.01	Affected	3.37	35	2	0	-1.0	34.02	246.4	-17.8	0.0	0.0	0.6	0.1						X		Potentially Benign	The iso-butyl side chain of Leu489, located in the α-helix (res. Leu489-Glu519) within an inter-helix space of four helices (res. Ala461-Phe476, res. Val441-Ser457, and res. Met414-Glu436), packs with hydrophobic residues (e.g., Cys432, Ala448, Lys444, Ala493, Val447, Met468) in the inter-helix space. In the variant simulations, the phenyl ring of the Phe489 side chain can also pack favorably in the hydrophobic region. However, due to the size difference, the aromatic side chain of Phe489 tends to reposition to escape the tight region to accommodate the larger side chain, stacking with Lys444. Although no apparent negative changes are observed during the variant simulation, the size difference between the swapped residues could affect the protein folding process.
c.1873C>G	L625V	Likely Pathogenic	GAP	Uncertain		1				-11.319	Likely Pathogenic	0.833	Likely Pathogenic	Ambiguous	0.480	Likely Benign	1.80	Ambiguous	0.7	1.69	Ambiguous	1.75	Ambiguous	1.42	Destabilizing	-2.96	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	3.07	Benign	0.01	Affected			2	1	0.4	-14.03
c.1544G>A	R515H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438787-G-A	3	1.86e-6	-10.774	Likely Pathogenic	0.337	Likely Benign	Likely Benign	0.730	Likely Pathogenic	1.07	Ambiguous	0.2	0.74	Ambiguous	0.91	Ambiguous	1.09	Destabilizing	-3.44	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.32	Pathogenic	0.01	Affected	3.37	35	2	0	1.3	-19.05	239.2	77.8	0.0	0.0	0.4	0.2		X						Potentially Benign	The guanidinium group of Arg515, located in the middle of an α-helix at the GAP domain (res. Gly502-Tyr518), forms salt bridges with the carboxylate groups of Glu512 on the same helix and Glu217 on a loop in the PH domain. Additionally, the positively charged Arg515 side chain forms hydrogen bonds with Leu610 and Gln612 in an opposing loop (res. Gly609-Asp616). In contrast, in the variant simulations, the imidazole ring of His515 cannot form salt bridges with either of the acidic residues, and its side chain is too short to form hydrogen bonds with the loop residues. Accordingly, the residue swap could weaken the tertiary structure assembly of the protein. Due to the missing N-terminal part of the SynGAP model, the effect could be largely underestimated or missing. Notably, the doubly protonated and positively charged form of histidine was not simulated here.
c.2050G>A	D684N (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.155	Likely Pathogenic	0.985	Likely Pathogenic	Likely Pathogenic	0.382	Likely Benign	1.47	Ambiguous	0.8	1.76	Ambiguous	1.62	Ambiguous	0.37	Likely Benign	-4.99	Deleterious	0.999	Probably Damaging	0.746	Possibly Damaging	3.39	Benign	0.01	Affected			2	1	0.0	-0.98
c.1652T>C	L551P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-14.620	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.953	Likely Pathogenic	6.66	Destabilizing	0.1	6.58	Destabilizing	6.62	Destabilizing	2.66	Destabilizing	-4.70	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.60	Pathogenic	0.01	Affected	3.37	35	-3	-3	-5.4	-16.04	208.6	60.9	0.1	0.0	-0.3	0.0	X							Potentially Pathogenic	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 pyrrolidine side chain of Pro551 is not as optimal as leucine for hydrophobic packing with the nearby residues. Moreover, Pro551 lacks the amide group, and thus, it cannot form a hydrogen bond with the backbone carbonyl group of Cys547, which disrupts the continuity of the secondary structure element.
c.2158G>A	D720N (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441623-G-A	5	3.10e-6	-9.135	Likely Pathogenic	0.654	Likely Pathogenic	Likely Benign	0.289	Likely Benign	0.01	Likely Benign	0.0	-0.20	Likely Benign	-0.10	Likely Benign	0.46	Likely Benign	-3.74	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	2.18	Pathogenic	0.01	Affected	3.50	9	1	2	0.0	-0.98
c.1715G>C	W572S (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-17.461	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.775	Likely Pathogenic	5.78	Destabilizing	0.2	3.37	Destabilizing	4.58	Destabilizing	1.79	Destabilizing	-12.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.24	Pathogenic	0.01	Affected	3.37	35	-2	-3	0.1	-99.14	235.1	76.6	0.0	0.0	-0.4	0.1							X	Potentially Pathogenic	The introduced residue Ser572, located in an α-helix (res. Arg563-Glu578), is considerably smaller than the tryptophan it replaced. The indole ring of the Trp572 side chain lies in a hydrophobic inter-helix space, where it makes extensive hydrophobic interactions with nearby residues such as Met470, Phe569, Leu588, and Ile589. In the variant simulations, all these favorable packing interactions are completely removed, as the introduced residue Ser572 is too hydrophilic or small to fill the hydrophobic niche occupied by the indole ring. Moreover, the hydroxyl group of Ser572 forms hydrogen bonds with the carbonyl groups of Glu567 and Val568 within the same α-helix, potentially lowering its integrity. Overall, the residue swap is highly likely to cause critical protein folding problems that are underestimated based on the effects seen in the variant simulations.
c.1718G>T	R573L (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-13.120	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.833	Likely Pathogenic	1.30	Ambiguous	0.6	1.11	Ambiguous	1.21	Ambiguous	0.80	Ambiguous	-5.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.41	Pathogenic	0.01	Affected	3.37	35	-3	-2	8.3	-43.03	237.4	60.7	0.0	0.0	-0.7	0.3	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 aliphatic iso-butyl group of the Leu573 side chain fails to establish any of these interactions, which, in turn, lowers the integrity of the opposing α-helix end (res. Glu582-Met603). Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.	10.1016/j.ajhg.2020.11.011
c.2214T>G	S738R	Likely Benign		Benign		1	6-33441679-T-G	1	6.20e-7	-4.241	Likely Benign	0.570	Likely Pathogenic	Likely Benign	0.068	Likely Benign										-1.55	Neutral	0.473	Possibly Damaging	0.193	Benign	2.69	Benign	0.01	Affected	4.32	2	0	-1	-3.7	69.11
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.2249G>A	G750E			Uncertain		1				-2.618	Likely Benign	0.413	Ambiguous	Likely Benign	0.146	Likely Benign										-2.27	Neutral	1.000	Probably Damaging	0.982	Probably Damaging	2.49	Pathogenic	0.01	Affected	3.99	5	0	-2	-3.1	72.06
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.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.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.1819C>G	L607V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33440871-C-G	2	1.24e-6	-11.190	Likely Pathogenic	0.637	Likely Pathogenic	Likely Benign	0.715	Likely Pathogenic	1.04	Ambiguous	0.2	1.36	Ambiguous	1.20	Ambiguous	0.90	Ambiguous	-2.99	Deleterious	0.985	Probably Damaging	0.992	Probably Damaging	-1.50	Pathogenic	0.01	Affected	3.37	35	2	1	0.4	-14.03	216.3	28.1	0.1	0.0	0.9	0.2				X				Potentially Benign	Leu607 is located in a short helical region (res. Ser606-Phe608) within an α-α loop connecting two α helices (res. Glu582-Met603 and res. Glu617-Asn635). In the WT simulations, the iso-butyl side chain of Leu607 does not interact with any other residues, but it could potentially interact directly with Ras due to its location at the GAP domain.In the variant simulations, Val607, which has similar size and physicochemical properties to leucine, does not cause any negative effects on the protein structure. However, due to its location at the GAP-Ras interface, the residue swap could affect the complex formation with the GTPase, but this cannot be investigated using solvent-only simulations.
c.2354G>A	R785H		SH3-binding motif	Uncertain		2	6-33442906-G-A	4	2.50e-6	-4.782	Likely Benign	0.388	Ambiguous	Likely Benign	0.129	Likely Benign										-2.61	Deleterious	0.999	Probably Damaging	0.947	Probably Damaging	2.25	Pathogenic	0.01	Affected	3.64	6	2	0	1.3	-19.05
c.2359C>A	P787T	Likely Pathogenic	SH3-binding motif	Likely Benign		1	6-33442911-C-A	17	1.05e-5	-4.813	Likely Benign	0.603	Likely Pathogenic	Likely Benign	0.258	Likely Benign										-4.40	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.46	Pathogenic	0.01	Affected	3.64	6	0	-1	0.9	3.99
c.1862G>A	R621Q (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33440914-G-A	19	1.18e-5	-14.682	Likely Pathogenic	0.910	Likely Pathogenic	Ambiguous	0.621	Likely Pathogenic	0.81	Ambiguous	0.1	1.13	Ambiguous	0.97	Ambiguous	1.35	Destabilizing	-3.98	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	2.82	Benign	0.01	Affected	3.37	35	1	1	1.0	-28.06	243.7	54.3	0.0	0.0	-0.4	0.2		X		X				Potentially Pathogenic	The guanidinium group of Arg621, located in an α helix (res. Glu617-Asn635), forms a salt bridge with Glu525 in a nearby loop and stacks with Leu635. In the variant simulations, the carboxamide side chain of Gln621, which can act as both a hydrogen bond acceptor and donor, also stacks with Leu635 but can only sporadically hydrogen bond with Glu525.Accordingly, the residue swap could affect the tertiary structure integrity by disrupting the salt bridge formation. Additionally, due to its location at the GAP-Ras interface, the residue swap could impact the complex formation with the GTPase, but this cannot be investigated using solvent-only simulations.
c.2420A>G	Y807C		SH3-binding motif	Uncertain		1	6-33442972-A-G	1	6.20e-7	-7.228	In-Between	0.204	Likely Benign	Likely Benign	0.243	Likely Benign										-3.89	Deleterious	0.997	Probably Damaging	0.934	Probably Damaging	2.42	Pathogenic	0.01	Affected	3.77	5	0	-2	3.8	-60.04
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.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.2548G>A	G850R	Likely Benign		Uncertain		1				-5.082	Likely Benign	0.398	Ambiguous	Likely Benign	0.194	Likely Benign										-0.07	Neutral	0.010	Benign	0.010	Benign	4.30	Benign	0.01	Affected	3.77	5	-3	-2	-4.1	99.14
c.2560C>T	R854C	Likely Benign		Uncertain		1	6-33443112-C-T	3	1.86e-6	-5.082	Likely Benign	0.170	Likely Benign	Likely Benign	0.174	Likely Benign										-2.48	Neutral	1.000	Probably Damaging	0.947	Probably Damaging	4.05	Benign	0.01	Affected	3.88	3	-3	-4	7.0	-53.05
c.2143C>T	P715S (3D Viewer)		GAP	Likely Pathogenic		1	6-33441608-C-T	1	6.20e-7	-7.635	In-Between	0.787	Likely Pathogenic	Ambiguous	0.277	Likely Benign	3.54	Destabilizing	0.0	0.81	Ambiguous	2.18	Destabilizing	0.94	Ambiguous	-7.17	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.43	Benign	0.01	Affected	3.50	9	1	-1	0.8	-10.04	231.8	-14.0	-0.1	0.0	-0.8	0.1							X	Uncertain	Pro715, along with Gly712 and Pro713, are located in a hinge region of an α-helix making a ~90-degree turn (res. Lys705-Leu725). In the WT simulations, the pyrrolidine side chain of Pro715, lacking the backbone amide groups altogether, forces the tight helix turn to take place while also hydrophobically packing with nearby residues (e.g., Leu700, Leu708, Leu714, and Leu718). Leu715, with a normal amide backbone, could potentially affect protein folding and turn formation, although this was not observed in the variant simulations. Additionally, the hydroxyl group of the Ser715 side chain can form hydrogen bonds with the backbone carbonyl group of Gly712 and disrupt the hydrophobic packing arrangement of the leucine residues from the neighboring α-helices, impacting the GAP domain tertiary assembly.
c.2681G>A	G894E	Likely Benign		Uncertain		1	6-33443233-G-A	6	3.72e-6	-5.377	Likely Benign	0.859	Likely Pathogenic	Ambiguous	0.180	Likely Benign										-2.07	Neutral	1.000	Probably Damaging	1.000	Probably Damaging	2.68	Benign	0.01	Affected	4.32	4	0	-2	-3.1	72.06
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.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.2713C>T	R905C			Conflicting		2	6-33443265-C-T	15	9.31e-6	-5.578	Likely Benign	0.723	Likely Pathogenic	Likely Benign	0.194	Likely Benign										-3.14	Deleterious	1.000	Probably Damaging	0.980	Probably Damaging	2.57	Benign	0.01	Affected	3.77	5	-4	-3	7.0	-53.05
c.2741A>T	D914V	Likely Benign		Uncertain		1				-4.260	Likely Benign	0.723	Likely Pathogenic	Likely Benign	0.187	Likely Benign										-2.24	Neutral	0.999	Probably Damaging	0.986	Probably Damaging	2.64	Benign	0.01	Affected	3.77	5	-3	-2	7.7	-15.96
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.2971G>A	G991R	Likely Benign		Conflicting		3	6-33443523-G-A	8	4.96e-6	-3.934	Likely Benign	0.411	Ambiguous	Likely Benign	0.102	Likely Benign										-1.20	Neutral	0.984	Probably Damaging	0.772	Possibly Damaging	4.11	Benign	0.01	Affected	4.32	2	-3	-2	-4.1	99.14
c.2987C>G	P996R	Likely Benign		Benign		1				-4.457	Likely Benign	0.141	Likely Benign	Likely Benign	0.040	Likely Benign										-1.04	Neutral	0.144	Benign	0.085	Benign	4.26	Benign	0.01	Affected	4.32	4	-2	0	-2.9	59.07
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.3022G>A	D1008N	Likely Benign		Likely Benign		1	6-33443574-G-A	3	1.86e-6	-4.045	Likely Benign	0.714	Likely Pathogenic	Likely Benign	0.128	Likely Benign										-2.15	Neutral	0.999	Probably Damaging	0.997	Probably Damaging	2.75	Benign	0.01	Affected	3.77	5	2	1	0.0	-0.98
c.3023A>G	D1008G			Uncertain		1	6-33443575-A-G	1	6.20e-7	-3.213	Likely Benign	0.742	Likely Pathogenic	Likely Benign	0.203	Likely Benign										-2.84	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.65	Benign	0.01	Affected	3.77	5	-1	1	3.1	-58.04
c.3026A>C	E1009A	Likely Pathogenic		Uncertain		1				-3.118	Likely Benign	0.679	Likely Pathogenic	Likely Benign	0.109	Likely Benign										-3.06	Deleterious	0.980	Probably Damaging	0.630	Possibly Damaging	2.39	Pathogenic	0.01	Affected	3.77	5	0	-1	5.3	-58.04
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.3056G>A	R1019H	Likely Benign		Conflicting		2	6-33443608-G-A	67	4.15e-5	-4.610	Likely Benign	0.258	Likely Benign	Likely Benign	0.122	Likely Benign										-1.95	Neutral	0.995	Probably Damaging	0.845	Possibly Damaging	2.39	Pathogenic	0.01	Affected	3.77	5	2	0	1.3	-19.05
c.3056G>T	R1019L	Likely Pathogenic		Uncertain		1	6-33443608-G-T	2	1.24e-6	-5.194	Likely Benign	0.752	Likely Pathogenic	Likely Benign	0.110	Likely Benign										-3.57	Deleterious	0.800	Possibly Damaging	0.573	Possibly Damaging	2.40	Pathogenic	0.01	Affected	3.77	5	-2	-3	8.3	-43.03
c.3119G>T	G1040V	Likely Pathogenic		Uncertain		1	6-33443671-G-T	4	2.48e-6	-3.453	Likely Benign	0.645	Likely Pathogenic	Likely Benign	0.774	Likely Pathogenic										-2.89	Deleterious	0.827	Possibly Damaging	0.456	Possibly Damaging	-0.74	Pathogenic	0.01	Affected	3.77	5	-1	-3	4.6	42.08
c.3184G>A	G1062R	Likely Benign		Conflicting		2	6-33443736-G-A	7	4.35e-6	-6.933	Likely Benign	0.353	Ambiguous	Likely Benign	0.403	Likely Benign										-0.34	Neutral	0.797	Possibly Damaging	0.139	Benign	4.10	Benign	0.01	Affected	4.32	2	-3	-2	-4.1	99.14
c.3209_3210delinsCA	R1070T	Likely Benign		Uncertain		1				-5.093	Likely Benign	0.860	Likely Pathogenic	Ambiguous												-2.35	Neutral	0.948	Possibly Damaging	0.507	Possibly Damaging	3.78	Benign	0.01	Affected	3.77	5	-1	-1	3.8	-55.08
c.3307C>T	R1103C			Uncertain		1	6-33443859-C-T	6	3.92e-6	-2.440	Likely Benign	0.246	Likely Benign	Likely Benign	0.140	Likely Benign										-3.01	Deleterious	0.996	Probably Damaging	0.787	Possibly Damaging	2.41	Pathogenic	0.01	Affected	3.77	5	-3	-4	7.0	-53.05
c.3308G>A	R1103H	Likely Benign		Benign/Likely benign		3	6-33443860-G-A	31	2.03e-5	-3.622	Likely Benign	0.156	Likely Benign	Likely Benign	0.116	Likely Benign										-1.97	Neutral	0.996	Probably Damaging	0.733	Possibly Damaging	2.49	Pathogenic	0.01	Affected	3.77	5	2	0	1.3	-19.05
c.3370G>A	G1124R			Conflicting		3	6-33443922-G-A	24	1.60e-5	-8.918	Likely Pathogenic	0.534	Ambiguous	Likely Benign	0.243	Likely Benign										-0.58	Neutral	0.002	Benign	0.002	Benign	4.81	Benign	0.01	Affected	3.77	5	-3	-2	-4.1	99.14
c.3632T>A	M1211K	Likely Pathogenic	Coiled-coil	Likely Benign		1				-9.013	Likely Pathogenic	0.662	Likely Pathogenic	Likely Benign	0.595	Likely Pathogenic										-2.95	Deleterious	0.987	Probably Damaging	0.979	Probably Damaging	5.59	Benign	0.01	Affected	3.77	5	0	-1	-5.8	-3.02
c.3661C>T	R1221W	Likely Pathogenic	Coiled-coil	Conflicting		3	6-33446653-C-T	1	6.20e-7	-10.938	Likely Pathogenic	0.651	Likely Pathogenic	Likely Benign	0.174	Likely Benign										-4.57	Deleterious	1.000	Probably Damaging	0.987	Probably Damaging	2.50	Benign	0.01	Affected	3.77	5	2	-3	3.6	30.03
c.3821G>A	R1274H			Likely Benign		1	6-33447869-G-A	4	2.58e-6	-5.259	Likely Benign	0.256	Likely Benign	Likely Benign	0.149	Likely Benign										-3.20	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	2.49	Pathogenic	0.01	Affected	3.77	5	0	2	1.3	-19.05
c.3824G>T	R1275L			Likely Benign		1	6-33447872-G-T	1	6.45e-7	-6.052	Likely Benign	0.446	Ambiguous	Likely Benign	0.117	Likely Benign										-4.04	Deleterious	0.800	Possibly Damaging	0.277	Benign	2.55	Benign	0.01	Affected	3.77	5	-3	-2	8.3	-43.03
c.3983G>C	R1328P	Likely Benign		Benign		1	6-33451857-G-C			-1.220	Likely Benign	0.466	Ambiguous	Likely Benign	0.060	Likely Benign										-2.01	Neutral	0.927	Possibly Damaging	0.452	Possibly Damaging	4.06	Benign	0.01	Affected	3.77	5	0	-2	2.9	-59.07
c.407G>A	R136Q			Benign		1	6-33432704-G-A	13	9.17e-6	-11.146	Likely Pathogenic	0.950	Likely Pathogenic	Ambiguous	0.190	Likely Benign										-2.26	Neutral	0.957	Probably Damaging	0.342	Benign	3.52	Benign	0.01	Affected	3.61	5	1	1	1.0	-28.06
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.505G>A	D169N			Uncertain		1				-10.713	Likely Pathogenic	0.761	Likely Pathogenic	Likely Benign	0.110	Likely Benign										-2.04	Neutral	0.079	Benign	0.052	Benign	4.07	Benign	0.01	Affected	3.74	4	2	1	0.0	-0.98
c.718G>A	D240N	Likely Pathogenic	PH	Uncertain		1				-12.942	Likely Pathogenic	0.755	Likely Pathogenic	Likely Benign	0.701	Likely Pathogenic	0.22	Likely Benign	0.9	0.47	Likely Benign	0.35	Likely Benign	0.37	Likely Benign	-4.37	Deleterious	0.993	Probably Damaging	0.984	Probably Damaging	5.88	Benign	0.01	Affected			2	1	0.0	-0.98
c.719A>G	D240G	Likely Pathogenic	PH	Uncertain		1				-12.825	Likely Pathogenic	0.951	Likely Pathogenic	Ambiguous	0.912	Likely Pathogenic	1.85	Ambiguous	0.1	2.72	Destabilizing	2.29	Destabilizing	0.24	Likely Benign	-6.19	Deleterious	0.993	Probably Damaging	0.984	Probably Damaging	5.79	Benign	0.01	Affected			1	-1	3.1	-58.04
c.680G>A	G227E (3D Viewer)	Likely Pathogenic	PH	Conflicting		2	6-33435531-G-A	3	1.86e-6	-9.186	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.792	Likely Pathogenic	2.56	Destabilizing	0.4	5.36	Destabilizing	3.96	Destabilizing	0.94	Ambiguous	-6.49	Deleterious	0.906	Possibly Damaging	0.360	Benign	5.72	Benign	0.01	Affected	3.43	12	0	-2	-3.1	72.06	237.7	-112.1	0.1	0.3	0.0	0.3		X				X		Uncertain	The introduced residue Glu227 is located in a β hairpin loop connecting two anti-parallel β sheet strands (res. Cys219-Thr224 and Thr228-Ala232). In the variant simulations, the carboxylate group of Glu227 frequently forms a salt bridge with the amino group of the neighboring residue Lys229. Despite this interaction, the integrity of the secondary structure element is not compromised. However, the β hairpins are potential nucleation sites during the initial stages of protein folding. Additionally, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.860A>C	D287A (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-14.686	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.484	Likely Benign	0.30	Likely Benign	0.1	-0.04	Likely Benign	0.13	Likely Benign	0.40	Likely Benign	-7.35	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.58	Pathogenic	0.01	Affected	3.38	23	-2	0	5.3	-44.01
c.700C>T	R234W (3D Viewer)	Likely Pathogenic	PH	Uncertain		1	6-33435551-C-T	3	1.86e-6	-12.625	Likely Pathogenic	0.947	Likely Pathogenic	Ambiguous	0.805	Likely Pathogenic	0.96	Ambiguous	0.3	0.69	Ambiguous	0.83	Ambiguous	0.13	Likely Benign	-5.52	Deleterious	0.997	Probably Damaging	0.803	Possibly Damaging	5.76	Benign	0.01	Affected	3.40	14	2	-3	3.6	30.03	262.8	39.6	-0.1	0.0	-0.2	0.2		X						Potentially Pathogenic	The guanidinium group of Arg234, located in a β-α loop between an anti-parallel β sheet strand (residues Gly227-Phe231) and an α helix (res. Ala236-Val250), forms a salt bridge with the carboxylate group of Glu238 in the α helix. Occasionally, it also bonds with the GAP domain residues Ser678 and Glu680. Thus, the positively charged Arg234 could contribute to the tertiary structure assembly between the PH and GAP domains. In contrast, the indole side chain of Trp234 in the variant is located on the protein surface in the variant simulations and is unable to form any interactions.
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.762G>C	K254N (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-13.306	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.757	Likely Pathogenic	0.73	Ambiguous	0.2	1.87	Ambiguous	1.30	Ambiguous	1.19	Destabilizing	-4.23	Deleterious	0.384	Benign	0.070	Benign	5.93	Benign	0.01	Affected	3.39	15	1	0	0.4	-14.07	215.3	-21.0	-1.0	1.7	0.2	0.3		X						Potentially Pathogenic	The amino group of Lys254, located in an α-β loop connecting the PH and C2 domains (res. Lys251-Arg258), forms salt bridges with the carboxylate groups of Glu244 and Asp684. Since the neutral carboxamide group of the Asn254 side chain cannot form salt bridges with acidic residues, the residue swap potentially weakens the tertiary structure assembly and/or influences the loop positioning. Regardless, in both the variant and WT simulations, all hydrogen bonds formed by the residue’s side chain were broken, and the residue rotated outwards. The partially α helical conformation of the loop, which extends to a nearby α helix (res. Met414-Asn426), is dynamic, making it unclear if the mutation affects it.
c.773G>A	R258H (3D Viewer)		C2	Benign/Likely benign		3	6-33437678-G-A	10	6.20e-6	-10.533	Likely Pathogenic	0.525	Ambiguous	Likely Benign	0.830	Likely Pathogenic	1.60	Ambiguous	0.6	1.00	Ambiguous	1.30	Ambiguous	1.47	Destabilizing	-4.06	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	5.77	Benign	0.01	Affected	3.39	15	2	0	1.3	-19.05	212.5	81.8	0.1	0.0	-0.5	0.2		X						Potentially Pathogenic	The guanidinium group of Arg258, located at the end of an α-β loop connecting the PH domain to the C2 domain (res. Lys251-Arg258), forms hydrogen bonds with the carboxamide groups of Asn727 and Asn729 side chains, as well as with the backbone carbonyl groups of Ala724, Leu725, and Asn727 in the WT simulations. Although the imidazole group of His258 can act as a hydrogen bond donor/acceptor, the swapped residue is unable to maintain an equally well-coordinated hydrogen bond network for linking the C2 and GAP domains in the variant simulations.
c.961C>T	R321C (3D Viewer)	Likely Pathogenic	C2	Conflicting		2	6-33437866-C-T	9	5.58e-6	-10.025	Likely Pathogenic	0.387	Ambiguous	Likely Benign	0.495	Likely Benign	0.57	Ambiguous	0.1	0.56	Ambiguous	0.57	Ambiguous	0.18	Likely Benign	-4.59	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.89	Pathogenic	0.01	Affected	3.38	23	-3	-4	7.0	-53.05
c.872A>G	Y291C (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-8.997	Likely Pathogenic	0.967	Likely Pathogenic	Likely Pathogenic	0.505	Likely Pathogenic	2.90	Destabilizing	0.4	3.51	Destabilizing	3.21	Destabilizing	1.35	Destabilizing	-7.37	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.76	Pathogenic	0.01	Affected	3.38	23	0	-2	3.8	-60.04	205.2	66.1	0.1	0.0	-0.4	0.4	X						X	Potentially Pathogenic	The phenol group of the Tyr291 side chain, located in an anti-parallel β sheet strand (res. Met289-Pro298), packs against hydrophobic residues of the C2 and PH domains (e.g., Leu317, Leu286, Leu284, Pro208, Val209). The phenol ring of Tyr291 also forms favorable Met-aromatic stacking with the methyl group of Met289. In the variant simulation, the thiol group of the Cys291 side chain is not as suitable for the hydrophobic inter-domain space as the phenol ring of Tyr291. Consequently, the structural unity of the PH domain is weakened and ultimately unfolds in the second simulation. Moreover, the residue swap might result in severe detrimental effects on the C2 domain structure and the C2-PH domain tertiary structure assembly during folding.
c.878G>C	R293P (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-16.275	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.497	Likely Benign	3.62	Destabilizing	0.4	9.06	Destabilizing	6.34	Destabilizing	0.47	Likely Benign	-6.43	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.45	Pathogenic	0.01	Affected	3.38	23	0	-2	2.9	-59.07	202.3	132.0	0.1	0.0	0.1	0.1	X	X		X				Potentially Pathogenic	The guanidinium group of the Arg293 side chain, located in an anti-parallel β sheet strand (res. Met289-Pro298), packs against the phenol ring of the Tyr281 side chain or forms a salt bridge with the carboxylate group of Glu283 on the outer side of the C2 domain. In the WT simulations, the positively charged side chain of arginine remains outside the hydrophobic C2 domain, resulting in a twist in the β strand. The backbone amide bond of Arg293 potentially maintains this twist by forming a hydrogen bond with the carbonyl group of His210 or the hydroxyl group of Ser211 in the anti-parallel β sheet.Although this twist is also maintained in the variant simulations, replacing the positively charged residue with proline, which lacks the backbone amide group altogether, causes the β strand to unfold. Because Arg293 is positioned at the C2 and PH domain interface, the residue swap could significantly impact the tertiary structure assembly. Notably, Arg293 is located at the SynGAP-Ras interface, and its role in complex formation cannot be fully understood through solvent-only simulations.
c.899C>T	S300F (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-10.222	Likely Pathogenic	0.353	Ambiguous	Likely Benign	0.117	Likely Benign	-0.29	Likely Benign	0.4	0.16	Likely Benign	-0.07	Likely Benign	0.04	Likely Benign	-2.66	Deleterious	0.975	Probably Damaging	0.596	Possibly Damaging	1.52	Pathogenic	0.01	Affected	3.47	19	-3	-2	3.6	60.10	233.6	-67.6	-0.1	0.0	0.4	0.2	X	X						Potentially Pathogenic	The hydroxyl group of the Ser300 side chain, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Met289-Pro298, res. Thr305-Asn315), hydrogen bonds with the guanidinium group of Arg299 and the backbone amide group and side chain of Ser302. Thus, in the WT simulations, it contributes to the β hairpin stability. In the variant simulations, the phenol ring of Phe300 cannot form any side chain-related hydrogen bonds, and Arg299 is moved away from its central hairpin loop position.β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant. Due to its location near the membrane surface, the residue swap could also affect the C2 loop dynamics and SynGAP-membrane association. However, this is beyond the scope of the solvent-only simulations to unravel.
c.928G>A	E310K (3D Viewer)	Likely Pathogenic	C2	Conflicting		4				-14.601	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.764	Likely Pathogenic	1.97	Ambiguous	1.2	3.66	Destabilizing	2.82	Destabilizing	1.02	Destabilizing	-3.68	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	1.19	Pathogenic	0.01	Affected	3.38	19	0	1	-0.4	-0.94	213.4	58.0	0.1	0.0	0.2	0.1		X						Potentially Pathogenic	The carboxylate group of Glu310, located in an anti-parallel β sheet strand (res. Thr305-Asn315), is ideally positioned to interact with the side chain hydroxyl and backbone amide groups of Thr295 on a twisted anti-parallel β strand (res. Met289-Arg299). Because the carboxylate group can also interact with the GAP domain residues (e.g., Gln612, Tyr614), Glu310 plays a key role in maintaining the tertiary assembly between the C2 and GAP domains. In the variant simulations, the amino group of the Lys310 side chain hydrogen bonds with the GAP domain residues and forms a salt bridge with Glu613. Although no apparent negative effects are seen due to the residue swap, it is possible that the loss of hydrogen bonding with the hydroxyl group of the Thr295 side chain causes problems during folding, potentially compromising the twisting of the β sheet.
c.968T>C	L323P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-12.507	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.762	Likely Pathogenic	3.39	Destabilizing	0.6	8.46	Destabilizing	5.93	Destabilizing	2.20	Destabilizing	-4.80	Deleterious	0.999	Probably Damaging	0.977	Probably Damaging	0.59	Pathogenic	0.01	Affected	4.29	398	-3	-3	-5.4	-16.04	201.9	68.2	0.0	0.1	0.6	0.3	X							Potentially Pathogenic	The iso-butyl side chain of Leu323, located at the beginning of an anti-parallel β sheet strand (res. Ala322-Asp330), packs against multiple hydrophobic leucine residues (e.g., Leu264, Leu266, Leu284, Leu286). In contrast, in the variant simulations, the less bulky cyclic five-membered pyrrolidine ring of Pro323 cannot fill the hydrophobic space as effectively as the branched hydrocarbon side chain of leucine. Notably, the backbone amide group of Leu323 forms a hydrogen bond with the backbone carbonyl group of Cys285. Pro323 cannot form this bond due to the absence of the backbone amide group, resulting in partial unfolding of the anti-parallel β sheet end in the variant simulations.
c.968T>G	L323R (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-14.568	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.692	Likely Pathogenic	3.75	Destabilizing	0.4	4.47	Destabilizing	4.11	Destabilizing	2.15	Destabilizing	-4.70	Deleterious	0.999	Probably Damaging	0.969	Probably Damaging	0.59	Pathogenic	0.01	Affected	3.39	22	-3	-2	-8.3	43.03	261.8	-61.6	-0.4	0.2	0.8	0.2	X		X				X	Potentially Pathogenic	The iso-butyl side chain of Leu323, located at the beginning of an anti-parallel β sheet strand (res. Ala322-Asp330), packs against multiple hydrophobic leucine residues (e.g., Leu264, Leu266, Leu284, Leu286). In contrast, in the variant simulations, the positively charged guanidinium group of the Arg323 side chain is unsuitable for the hydrophobic niche. Consequently, the side chain either rotates away from the center of the C2 domain or, if it remains within the C2 domain core, it reorients nearby residues to form hydrogen bonds. Regardless, the residue swap extensively disrupts the C2 domain structure.
c.980T>C	L327P (3D Viewer)	Likely Pathogenic	C2	Pathogenic		3				-16.602	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.658	Likely Pathogenic	5.38	Destabilizing	0.1	4.00	Destabilizing	4.69	Destabilizing	2.62	Destabilizing	-5.97	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.52	Pathogenic	0.01	Affected	3.38	23	-3	-3	-5.4	-16.04	221.7	69.4	0.1	0.0	0.6	0.1	X							Potentially Pathogenic	The backbone amide group of Leu327, located in the middle of an anti-parallel β sheet strand (res. Ala322-Asp330), forms a hydrogen bond with the carbonyl group of Gly344 on a neighboring β strand (res. Lys336-Pro349) in the WT simulations. In contrast, in the variant simulations, the introduction of Pro327 destabilizes the hydrogen bonding between the two anti-parallel β strands because proline lacks the backbone amide group altogether. Additionally, in the WT simulations, the iso-butyl side chain of Leu327 packs against multiple hydrophobic residues (e.g., Leu274, V400, Val343), whereas the less bulky cyclic five-membered pyrrolidine ring of Pro327 cannot fill the same space as effectively. Thus, although no large-scale unfolding is observed during the variant simulations, the residue swap is likely to cause severe problems for the correct C2 domain folding, which could also affect the SynGAP-membrane association.	10.1016/j.ajhg.2020.11.011
c.1025A>G	Y342C (3D Viewer)	Likely Pathogenic	C2	Benign/Likely benign		2	6-33437930-A-G	21	1.30e-5	-7.596	In-Between	0.682	Likely Pathogenic	Likely Benign	0.404	Likely Benign	2.48	Destabilizing	0.1	2.73	Destabilizing	2.61	Destabilizing	0.92	Ambiguous	-6.67	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.72	Pathogenic	0.02	Affected	3.37	25	0	-2	3.8	-60.04	242.4	62.8	0.1	0.0	-0.1	0.2								Potentially Pathogenic	The phenol ring of Tyr342, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), faces outward in the C2 domain. This phenol ring contributes to a triple tyrosine stack (Tyr342, Tyr328, and Tyr281) that links together three anti-parallel β sheet strands. Additionally, it shields Gly344 from the solvent, reducing its exposure and providing stability for the β-sandwich. This motif also contributes to a twist formation in the β sheet.In the variant simulations, the Cys342 side chain cannot participate in the stack formation. Instead, its thiol group forms a hydrogen bond with the backbone carbonyl group of Leu327. Although these changes in surface interactions could weaken the characteristic twist that strengthens the β sheet fold, no major structural effects are observed in the variant simulations. The residue swap could also affect the SynGAP-membrane association; however, this phenomenon cannot be addressed using solvent-only simulations. Notably, the thiol group of cysteine is not a particularly strong hydrogen-bonding partner, which could mitigate the negative effects of the residue swap.
c.1058T>C	L353P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-7.913	In-Between	0.936	Likely Pathogenic	Ambiguous	0.464	Likely Benign	4.63	Destabilizing	0.1	10.19	Destabilizing	7.41	Destabilizing	2.17	Destabilizing	-3.70	Deleterious	0.947	Possibly Damaging	0.454	Possibly Damaging	1.29	Pathogenic	0.02	Affected	3.37	25	-3	-3	-5.4	-16.04
c.1213C>T	R405C (3D Viewer)	Likely Pathogenic	C2	Conflicting		2	6-33438118-C-T	6	3.72e-6	-9.206	Likely Pathogenic	0.713	Likely Pathogenic	Likely Benign	0.427	Likely Benign	0.72	Ambiguous	0.1	1.51	Ambiguous	1.12	Ambiguous	1.21	Destabilizing	-7.27	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.61	Benign	0.02	Affected	3.38	28	-4	-3	7.0	-53.05	221.3	82.6	-0.1	0.0	-0.2	0.3	X	X						Potentially Pathogenic	The guanidinium group of Arg405, located in an anti-parallel β sheet strand of the C2 domain (res. Ala399-Ile411), forms a salt bridge with the carboxylate group of the Glu446 side chain from an opposing α helix (res. Val441-Ser457) in the GAP domain. The positively charged Arg405 side chain also stacks with the aromatic ring of the Phe358 side chain from a loop preceding the β strand (res. Thr359-Thr366), which could assist in maintaining the anti-parallel strand arrangement.In the variant simulations, the thiol-containing side chain of Cys405 is neutral and smaller compared to the arginine side chain. The lack of Arg405-Phe358 stacking affects the loop structure, causing it to assume a β strand form—an effect that could be exacerbated during protein folding. Moreover, the inability of Cys405 to form a salt bridge with Glu446 could affect the tertiary structure assembly, although this is not apparent based on the variant simulations.
c.1484A>G	E495G (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438516-A-G	1	6.20e-7	-9.400	Likely Pathogenic	0.923	Likely Pathogenic	Ambiguous	0.867	Likely Pathogenic	1.21	Ambiguous	0.0	2.06	Destabilizing	1.64	Ambiguous	0.78	Ambiguous	-6.70	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.46	Pathogenic	0.02	Affected	3.37	35	-2	0	3.1	-72.06
c.1256A>G	E419G (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.589	Likely Pathogenic	0.956	Likely Pathogenic	Likely Pathogenic	0.469	Likely Benign	1.41	Ambiguous	0.0	1.94	Ambiguous	1.68	Ambiguous	0.83	Ambiguous	-6.42	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	3.31	Benign	0.02	Affected	3.37	29	0	-2	3.1	-72.06	165.3	110.8	0.0	0.0	-0.1	0.0		X						Potentially Pathogenic	The carboxylate group of Glu419, located on an α helix (res. Met414-Glu436), forms a salt bridge with the side chain of either Arg716 or Lys418 from an opposing helix (res. Pro713-Arg726). The backbone amide group of Glu419 does not form H-bonds, resulting in a slight bend in the α helix. Thus, although glycine is known as an “α helix breaker,” the residue swap does not disrupt the continuity or integrity of the α helix. However, because Gly419 cannot form a salt bridge with the guanidinium group of the Arg716 side chain, the C2-GAP domain tertiary structure could be compromised during folding.
c.1306G>A	E436K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.869	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.829	Likely Pathogenic	0.56	Ambiguous	0.1	2.86	Destabilizing	1.71	Ambiguous	0.82	Ambiguous	-3.77	Deleterious	0.994	Probably Damaging	0.951	Probably Damaging	4.71	Benign	0.02	Affected	3.37	29	0	1	-0.4	-0.94	186.8	39.8	0.0	0.0	-0.2	0.0	X	X		X				Potentially Pathogenic	The carboxylate group of Glu436, located on the α helix (res. Met414-Glu436), forms a salt bridge with the amino group of the Lys444 side chain on an opposing α helix (res. Val441-Ser457). The backbone carbonyl of Glu436 also H-bonds with the Lys444 side chain, which helps keep the ends of the two α helices tightly connected. In contrast, in the variant simulations, the salt bridge formation with Lys444 is not possible. Instead, the repelled Lys436 side chain rotates outward, causing a change in the α helix backbone H-bonding: the amide group of Lys444 H-bonds with the carbonyl of Ala433 instead of the carbonyl of Cys432.
c.1667A>T	N556I (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438910-A-T			-13.391	Likely Pathogenic	0.929	Likely Pathogenic	Ambiguous	0.761	Likely Pathogenic	0.64	Ambiguous	0.0	0.17	Likely Benign	0.41	Likely Benign	0.26	Likely Benign	-7.52	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.35	Pathogenic	0.02	Affected	3.37	35	-3	-2	8.0	-0.94
c.1792C>G	L598V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.002	Likely Pathogenic	0.578	Likely Pathogenic	Likely Benign	0.221	Likely Benign	1.89	Ambiguous	0.1	1.58	Ambiguous	1.74	Ambiguous	1.01	Destabilizing	-2.92	Deleterious	0.944	Possibly Damaging	0.786	Possibly Damaging	3.21	Benign	0.02	Affected	3.37	35	2	1	0.4	-14.03	218.4	29.6	0.0	0.0	0.8	0.0						X		Potentially Benign	The iso-butyl side chain of Leu598, located on an α helix (res. Glu582-Met603), packs hydrophobically with other hydrophobic residues in the inter-helix space (e.g., Ile602, Phe594, Ile510).In the variant simulations, Val598, which has similar size and physicochemical properties to leucine, resides in the inter-helix hydrophobic space in a similar manner to Leu598 in the WT. This causes no negative effects on the protein structure.
c.1487A>G	E496G (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.529	Likely Pathogenic	0.850	Likely Pathogenic	Ambiguous	0.825	Likely Pathogenic	1.83	Ambiguous	0.1	1.76	Ambiguous	1.80	Ambiguous	0.92	Ambiguous	-6.16	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.45	Pathogenic	0.02	Affected	3.37	35	0	-2	3.1	-72.06	173.9	103.1	0.0	0.0	-0.7	0.0		X		X				Potentially Pathogenic	Glu496 is located in the α-helix (res. Leu489-Glu519), and its carboxylate group forms salt bridges with the neighbouring residues Lys492 and Arg499 in the WT simulations. Glu496 also forms a hydrogen bond with Ser449 on an opposing helix (res. Val441-Ser457). In the variant simulations, Gly496 cannot form these salt bridges, which could weaken the secondary structure. Additionally, the loss of the hydrogen bond with Ser449 on the opposite helix can weaken the tertiary structure assembly. Moreover, glycine is an α-helix breaker, and it is seen to weaken the integrity of the helix as the hydrogen bonding between the backbone atoms of Gly496 and Ala493 breaks down. Also, due to its location at the GAP-Ras interface, the interaction of Glu496 with Arg499 and Lys492 might play a role in complex association and stability, which cannot be fully addressed using the SynGAP solvent-only simulations.
c.1639T>C	C547R (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.967	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.900	Likely Pathogenic	7.76	Destabilizing	0.8	5.83	Destabilizing	6.80	Destabilizing	1.69	Destabilizing	-11.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.33	Pathogenic	0.02	Affected	3.37	35	-4	-3	-7.0	53.05	267.4	-90.3	0.0	0.0	-0.1	0.1	X	X	X				X	Potentially Pathogenic	Cys547 is located in an α-helix (res. Ala533-Val560). The thiol side chain of Cys is situated in a hydrophobic inter-helix space, where it packs hydrophobically with other residues such as Ile626, Leu551, and Phe652. Additionally, the thiol side chain of Cys547 weakly hydrogen bonds with the carbonyl group of Leu543 in the same α-helix. In the variant simulations, the bulkier, positively charged guanidinium group of Arg547 must rotate out of the hydrophobic space. Consequently, it forms ionic interactions with the carboxylate groups of Glu548 in the same helix and Glu656 in the neighboring α-helix (res. Glu666-Asp644). This causes the two helices to slightly separate, significantly affecting the secondary structure integrity of the latter helix. These negative structural effects could be more pronounced during protein folding and are likely to be undermined in the MD simulations.
c.2115G>C	K705N (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-9.767	Likely Pathogenic	0.925	Likely Pathogenic	Ambiguous	0.183	Likely Benign	0.74	Ambiguous	0.0	0.37	Likely Benign	0.56	Ambiguous	0.44	Likely Benign	-3.12	Deleterious	0.996	Probably Damaging	0.876	Possibly Damaging	3.37	Benign	0.02	Affected	3.47	10	1	0	0.4	-14.07	221.4	-20.2	0.0	0.0	0.0	0.1						X		Uncertain	The amino side chain of Lys705, located at the end and outer surface of an α-helix (res. Thr704-Gly712), does not form any interactions in the WT simulations. In the variant simulations, the carboxamide side chain of Asn705 briefly forms a salt bridge with Glu706. However, there is no apparent difference between the systems. Due to the model ending abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.1723C>T	R575C (3D Viewer)	Likely Pathogenic	GAP	Conflicting		3	6-33440775-C-T	23	1.43e-5	-11.179	Likely Pathogenic	0.630	Likely Pathogenic	Likely Benign	0.715	Likely Pathogenic	1.39	Ambiguous	0.2	0.50	Ambiguous	0.95	Ambiguous	0.73	Ambiguous	-5.43	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.30	Pathogenic	0.02	Affected	3.37	35	-4	-3	7.0	-53.05	227.7	99.2	0.0	0.0	0.0	0.1		X						Potentially Pathogenic	The guanidinium group of Arg575, located in an α-helix (res. Arg563-Glu578), forms salt bridges with the carboxylate groups of Asp463 and Asp467, and it also hydrogen bonds with the hydroxyl group of Ser466 on an opposing α-helix (res. Ala461-Phe476) in the WT simulations. In the variant simulations, the thiol group of the Cys575 side chain, which is neither positively charged nor particularly hydrophilic, packs against the hydrophobic Met470 on an opposing α-helix (res. Ala461-Arg475). Additionally, although the thiol group is not an effective hydrogen bonder, the Cys575 side chain rotates to hydrogen bond with the backbone carbonyl group of Ser571 in the same α-helix, which could theoretically lower the helix integrity. Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.
c.2243T>G	L748R	Likely Benign		Conflicting		2	6-33441708-T-G	3	1.86e-6	-3.331	Likely Benign	0.245	Likely Benign	Likely Benign	0.055	Likely Benign										-0.67	Neutral	0.912	Possibly Damaging	0.448	Possibly Damaging	2.73	Benign	0.02	Affected	4.32	2	-3	-2	-8.3	43.03
c.2305C>T	L769F	Likely Benign		Uncertain		1				-5.044	Likely Benign	0.146	Likely Benign	Likely Benign	0.060	Likely Benign										-0.89	Neutral	0.925	Possibly Damaging	0.510	Possibly Damaging	3.94	Benign	0.02	Affected			2	0	-1.0	34.02
c.2359C>T	P787S		SH3-binding motif	Uncertain		1	6-33442911-C-T	3	1.86e-6	-4.203	Likely Benign	0.564	Ambiguous	Likely Benign	0.221	Likely Benign										-3.81	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.48	Pathogenic	0.02	Affected	3.64	6	-1	1	0.8	-10.04
c.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.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.1947G>C	M649I (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.361	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.449	Likely Benign	2.42	Destabilizing	0.2	1.96	Ambiguous	2.19	Destabilizing	1.01	Destabilizing	-3.99	Deleterious	0.672	Possibly Damaging	0.093	Benign	3.40	Benign	0.02	Affected	3.38	27	2	1	2.6	-18.03	243.7	21.5	0.0	0.1	0.0	0.1							X	Potentially Benign	The thioether side chain of Met649, located on an α helix (res. Ser641-Glu666), bridges Phe652, Phe648, and Phe639 in an inter-helix hydrophobic cavity in the WT simulations. In the variant simulations, the sec-butyl side chain of Ile649 maintains hydrophobic interactions with nearby residues, with no significant effects on the protein structure.However, methionine is known as a bridging motif for aromatic residues, and these Met-aromatic interactions are lost in the variant. Indeed, in the second variant simulation,the bridging of Phe652, Phe648 and Phe639 is completely lost. In reality, the effect could be more severe on the structure during the protein folding.
c.1966G>C	E656Q (3D Viewer)		GAP	Uncertain		1	6-33441225-G-C	1	6.20e-7	-9.145	Likely Pathogenic	0.766	Likely Pathogenic	Likely Benign	0.249	Likely Benign	-0.14	Likely Benign	0.0	-0.81	Ambiguous	-0.48	Likely Benign	0.25	Likely Benign	-2.29	Neutral	0.980	Probably Damaging	0.528	Possibly Damaging	3.46	Benign	0.02	Affected	3.39	24	2	2	0.0	-0.98	224.3	1.7	0.0	0.1	0.1	0.0		X						Potentially Benign	The carboxylate side chain of Glu656, located on an α helix (res. Ser641-Glu666), frequently forms a hydrogen bond with the nearby residue Ser659 on the same α helix. In the variant simulations, the carboxamide side chain of Gln656 alternatively forms a hydrogen bond with either Ser659 or Glu548 on an opposing helix (res. Ala533-Val560).Although the frequent interaction between Gln656 and Glu548 may strengthen or stabilize the tertiary structure assembly, the effect is likely to be marginal.
c.1997A>G	E666G (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441256-A-G	10	6.20e-6	-12.261	Likely Pathogenic	0.911	Likely Pathogenic	Ambiguous	0.522	Likely Pathogenic	1.57	Ambiguous	0.1	1.46	Ambiguous	1.52	Ambiguous	0.93	Ambiguous	-6.25	Deleterious	1.000	Probably Damaging	0.970	Probably Damaging	3.37	Benign	0.02	Affected	3.38	28	0	-2	3.1	-72.06	173.9	98.5	0.0	0.0	-0.7	0.0		X						Potentially Pathogenic	In the WT simulations, the carboxylate group of Glu666, located on the α-helix (res. Ser641-Glu666), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), such as Lys566, Thr672, and Asn669. In the variant simulations, the carbonyl group of Gly666 occasionally forms hydrogen bonds with Lys566 and Asn669. However, Gly666 lacks a side chain and thus cannot maintain as well-coordinated a hydrogen-bond network as Glu666 in the WT, which may affect the tertiary structure assembly.
c.2443C>A	R815S		SH3-binding motif	Benign		1				-7.324	In-Between	0.950	Likely Pathogenic	Ambiguous	0.138	Likely Benign										-1.86	Neutral	0.999	Probably Damaging	0.997	Probably Damaging	2.67	Benign	0.02	Affected			0	-1	3.7	-69.11
c.2443C>G	R815G		SH3-binding motif	Uncertain		1				-7.983	In-Between	0.854	Likely Pathogenic	Ambiguous	0.146	Likely Benign										-3.22	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.62	Benign	0.02	Affected	4.32	4	-3	-2	4.1	-99.14
c.2444G>A	R815H		SH3-binding motif	Likely Benign		2	6-33442996-G-A	24	1.49e-5	-7.474	In-Between	0.553	Ambiguous	Likely Benign	0.157	Likely Benign										-1.81	Neutral	1.000	Probably Damaging	0.998	Probably Damaging	2.61	Benign	0.02	Affected	4.32	4	2	0	1.3	-19.05																10.1016/j.ajhg.2020.11.011
c.2521G>A	V841M			Uncertain		1	6-33443073-G-A	3	1.86e-6	-7.000	In-Between	0.651	Likely Pathogenic	Likely Benign	0.119	Likely Benign										-0.74	Neutral	0.999	Probably Damaging	0.998	Probably Damaging	2.54	Benign	0.02	Affected	3.77	5	1	2	-2.3	32.06
c.2089T>C	W697R (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441348-T-C	1	6.20e-7	-10.020	Likely Pathogenic	0.941	Likely Pathogenic	Ambiguous	0.401	Likely Benign	1.14	Ambiguous	0.1	1.18	Ambiguous	1.16	Ambiguous	1.25	Destabilizing	-9.50	Deleterious	1.000	Probably Damaging	0.994	Probably Damaging	3.45	Benign	0.02	Affected	3.46	13	2	-3	-3.6	-30.03	254.4	-41.2	0.0	0.0	-0.7	0.0						X		Potentially Benign	The indole ring of Trp697, located on the outer surface of an α-helix (res. Leu685-Val699), is not involved in any long-lasting interactions in the WT simulations. In the variant simulations, the positively charged guanidinium side chain of Arg697 occasionally forms hydrogen bonds with nearby residues, such as Ser722 and Asn719. However, similar to Trp697 in the WT, Arg697 does not form any long-lasting interactions and thus does not induce any negative structural effects in the simulations.
c.2522T>C	V841A			Uncertain		1	6-33443074-T-C	3	1.86e-6	-8.152	Likely Pathogenic	0.901	Likely Pathogenic	Ambiguous	0.183	Likely Benign										-2.13	Neutral	0.992	Probably Damaging	0.989	Probably Damaging	2.57	Benign	0.02	Affected	3.77	5	0	0	-2.4	-28.05
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.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.2147G>A	R716Q (3D Viewer)		GAP	Conflicting		2	6-33441612-G-A	4	2.48e-6	-8.338	Likely Pathogenic	0.308	Likely Benign	Likely Benign	0.210	Likely Benign	-0.01	Likely Benign	0.0	0.47	Likely Benign	0.23	Likely Benign	0.58	Ambiguous	-3.14	Deleterious	1.000	Probably Damaging	0.990	Probably Damaging	3.35	Benign	0.02	Affected	3.50	9	1	1	1.0	-28.06	250.0	48.9	0.0	0.0	-0.5	0.0						X		Uncertain	The guanidinium group of Arg716, located on the outer surface of an α-helix (res. Leu714-Arg726), forms a salt bridge with the carboxylate group of Asp720. In the variant simulations, the carboxamide group of Gln716 also forms a hydrogen bond with the carboxylate group of Asp720, although this bond is weaker than the Arg716 salt bridge in the WT. Overall, no adverse effects on the protein structure are observed in the simulations. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
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.2855G>T	G952V	Likely Benign		Uncertain		1				-7.074	In-Between	0.078	Likely Benign	Likely Benign	0.231	Likely Benign										-0.33	Neutral	0.000	Benign	0.000	Benign	3.20	Benign	0.02	Affected	3.77	5	-1	-3	4.6	42.08
c.3254G>A	R1085Q	Likely Benign		Uncertain		1	6-33443806-G-A	5	3.16e-6	-3.843	Likely Benign	0.589	Likely Pathogenic	Likely Benign	0.224	Likely Benign										-1.43	Neutral	0.998	Probably Damaging	0.988	Probably Damaging	2.73	Benign	0.02	Affected	3.77	5	1	1	1.0	-28.06
c.3308G>T	R1103L	Likely Benign		Uncertain		1	6-33443860-G-T			-2.330	Likely Benign	0.205	Likely Benign	Likely Benign	0.173	Likely Benign										-2.35	Neutral	0.002	Benign	0.005	Benign	2.44	Pathogenic	0.02	Affected	3.77	5	-3	-2	8.3	-43.03
c.3313C>T	R1105W			Uncertain		1	6-33443865-C-T	6	3.93e-6	-6.911	Likely Benign	0.488	Ambiguous	Likely Benign	0.133	Likely Benign										-4.34	Deleterious	0.999	Probably Damaging	0.696	Possibly Damaging	2.42	Pathogenic	0.02	Affected	3.77	5	-3	2	3.6	30.03
c.3376G>T	G1126C	Likely Benign		Uncertain		1	6-33443928-G-T	11	7.35e-6	-9.389	Likely Pathogenic	0.113	Likely Benign	Likely Benign	0.449	Likely Benign										-1.40	Neutral	0.005	Benign	0.005	Benign	4.74	Benign	0.02	Affected	3.77	5	-3	-3	2.9	46.09
c.3377G>A	G1126D			Uncertain		1				-8.888	Likely Pathogenic	0.432	Ambiguous	Likely Benign	0.376	Likely Benign										-0.65	Neutral	0.906	Possibly Damaging	0.473	Possibly Damaging	4.82	Benign	0.02	Affected	3.77	5	1	-1	-3.1	58.04
c.3511_3512delinsTG	A1171C	Likely Benign	Coiled-coil	Uncertain		1				-5.363	Likely Benign	0.496	Ambiguous	Likely Benign												-1.16	Neutral	0.978	Probably Damaging	0.825	Possibly Damaging	5.32	Benign	0.02	Affected	4.32	4	-2	0	0.7	32.06
c.373C>T	P125S	Likely Benign		Uncertain		1				-3.769	Likely Benign	0.238	Likely Benign	Likely Benign	0.121	Likely Benign										-3.57	Deleterious	0.580	Possibly Damaging	0.140	Benign	2.86	Benign	0.02	Affected	3.61	5	1	-1	0.8	-10.04
c.380G>A	R127Q	Likely Benign		Uncertain		1	6-33432245-G-A	6	3.72e-6	-1.711	Likely Benign	0.320	Likely Benign	Likely Benign	0.037	Likely Benign										-1.04	Neutral	0.006	Benign	0.001	Benign	4.04	Benign	0.02	Affected	3.74	4	1	1	1.0	-28.06
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.4013G>A	R1338Q	Likely Benign		Conflicting		3	6-33451887-G-A	12	8.40e-6	-3.494	Likely Benign	0.317	Likely Benign	Likely Benign	0.076	Likely Benign										-1.87	Neutral	0.896	Possibly Damaging	0.194	Benign	3.81	Benign	0.02	Affected	3.77	5	1	1	1.0	-28.06
c.404G>A	R135Q			Uncertain		1	6-33432701-G-A	5	3.84e-6	-8.011	Likely Pathogenic	0.853	Likely Pathogenic	Ambiguous	0.087	Likely Benign										-1.94	Neutral	0.327	Benign	0.100	Benign	3.76	Benign	0.02	Affected	3.61	5	1	1	1.0	-28.06
c.667A>T	T223S (3D Viewer)		PH	Conflicting		2	6-33435518-A-T	3	1.86e-6	-7.714	In-Between	0.410	Ambiguous	Likely Benign	0.535	Likely Pathogenic	0.26	Likely Benign	0.1	0.50	Ambiguous	0.38	Likely Benign	0.62	Ambiguous	-2.86	Deleterious	0.421	Benign	0.058	Benign	5.80	Benign	0.02	Affected	3.41	13	1	1	-0.1	-14.03	200.7	17.3	-0.2	0.2	0.0	0.0						X		Uncertain	The introduced residue Ser223 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Its hydroxyl group forms hydrogen bonds with nearby residues Thr228 and Lys207 in the variant simulations, similar to the hydroxyl group of Thr223 in the WT simulations. These hydrogen-bonding interactions at the β sheet surface contribute to the stability of the secondary structure element and may prevent it from unfolding. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.767A>G	N256S (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-10.640	Likely Pathogenic	0.950	Likely Pathogenic	Ambiguous	0.707	Likely Pathogenic	0.31	Likely Benign	0.2	0.36	Likely Benign	0.34	Likely Benign	0.48	Likely Benign	-4.33	Deleterious	0.997	Probably Damaging	0.970	Probably Damaging	5.87	Benign	0.02	Affected	3.39	15	1	1	2.7	-27.03
c.937G>A	E313K (3D Viewer)	Likely Pathogenic	C2	Likely Benign		1				-12.902	Likely Pathogenic	0.959	Likely Pathogenic	Likely Pathogenic	0.575	Likely Pathogenic	0.64	Ambiguous	0.6	1.40	Ambiguous	1.02	Ambiguous	0.75	Ambiguous	-3.31	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	1.90	Pathogenic	0.02	Affected			0	1	-0.4	-0.94
c.896G>A	R299H (3D Viewer)		C2	Conflicting		2	6-33437801-G-A	10	6.20e-6	-7.731	In-Between	0.388	Ambiguous	Likely Benign	0.238	Likely Benign	3.97	Destabilizing	1.0	0.94	Ambiguous	2.46	Destabilizing	1.41	Destabilizing	-3.35	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.69	Pathogenic	0.02	Affected	3.39	19	2	0	1.3	-19.05	211.2	72.5	-0.1	0.2	-0.2	0.3	X							Potentially Pathogenic	The guanidinium group of Arg299, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Met289-Pro298, res. Thr305-Asn315), forms hydrogen bonds that stabilize the tight turn. In the WT simulations, the Arg299 side chain hydrogen bonds with the loop backbone carbonyl groups (e.g., Ser302, Thr305, Leu274, Gly303), the hydroxyl group of Ser300, and even forms a salt bridge with the carboxylate group of Asp304.In the variant simulations, the imidazole ring of His299 (epsilon protonated state) hydrogen bonds with the carbonyl group of Asp304 and the hydroxyl group of Ser300. However, it does not form as many or as strong interactions as arginine, which could affect the initial formation of the secondary hairpin loop during folding. β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant.Additionally, His299 prefers to hydrophobically interact with other hydrophobic residues inside the C2 domain core (e.g., Val306, Leu274), which destabilizes the C2 domain. Indeed, the β strand partially unfolds during the second simulation. Moreover, the positively charged Arg299 side chain faces the polar head group region of the inner leaflet membrane and could directly anchor the C2 domain to the membrane. In short, the residue swap could negatively affect both protein folding and the stability of the SynGAP-membrane association.
c.930G>C	E310D (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-11.218	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.666	Likely Pathogenic	1.87	Ambiguous	0.5	2.39	Destabilizing	2.13	Destabilizing	1.04	Destabilizing	-2.76	Deleterious	0.997	Probably Damaging	0.992	Probably Damaging	1.19	Pathogenic	0.02	Affected	3.38	19	3	2	0.0	-14.03	232.6	27.2	0.1	0.0	0.1	0.1						X		Potentially Benign	The carboxylate group of Glu310, located in an anti-parallel β sheet strand (res. Thr305-Asn315), is ideally positioned to interact with the hydroxyl and backbone amide groups of Thr295 on a twisted anti-parallel β strand. Because the carboxylate group can also interact with the GAP domain residues (e.g., Gln612, Tyr614), Glu310 potentially plays a key role in maintaining the tertiary assembly between the C2 and GAP domains. In the variant simulations, the carboxylate group of Asp310 can form the same interactions as glutamate; however, due to its one hydrocarbon shorter length, the connections are less stable or less optimal.
c.1004G>A	R335H (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437909-G-A	2	1.24e-6	-12.521	Likely Pathogenic	0.831	Likely Pathogenic	Ambiguous	0.132	Likely Benign	0.58	Ambiguous	0.1	0.22	Likely Benign	0.40	Likely Benign	0.72	Ambiguous	-3.02	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.70	Pathogenic	0.03	Affected	3.38	22	2	0	1.3	-19.05	242.4	82.1	-2.4	0.6	-0.1	0.1								Uncertain	The guanidinium group of Arg335, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Ala322-Asp330, res. Gly341-Pro349), faces the post-synaptic inner membrane surface. In the WT simulations, the Arg335 side chain dynamically forms salt bridges with the carboxylate groups of Asp322, Asp338, and Asp616. In contrast, the imidazole ring of His335, which is not double protonated and thus not positively charged in the variant simulations, continues to move dynamically without forming any lasting or strong interactions. Importantly, the positively charged arginine residues of the C2 domain are ideal membrane anchors for ensuring SynGAP-membrane association. However, this phenomenon cannot be addressed using solvent-only simulations.
c.1042G>A	V348M (3D Viewer)		C2	Uncertain		1				-7.076	In-Between	0.546	Ambiguous	Likely Benign	0.191	Likely Benign	-1.19	Ambiguous	0.1	0.72	Ambiguous	-0.24	Likely Benign	0.76	Ambiguous	-1.62	Neutral	0.966	Probably Damaging	0.564	Possibly Damaging	1.58	Pathogenic	0.03	Affected	3.37	25	2	1	-2.3	32.06	253.8	-47.4	-0.3	0.1	0.2	0.1						X		Potentially Benign	The iso-propyl side chain of Val348, located in an anti-parallel β sheet strand (res. Gly341-Pro349), packs against multiple hydrophobic C2 domain residues (e.g., Leu353, Leu323, Leu402). In the variant simulations, the thioether side chain of Met348 can form similar interactions as valine due to its comparable hydrophobic profile. In fact, the thioether group of methionine can even stack favorably with the phenol ring of Tyr363 in the anti-parallel β sheet strand (res. Ala399-Ile411). Overall, the residue swap does not appear to cause negative effects on the protein structure based on the simulations.
c.1447A>G	I483V (3D Viewer)		GAP	Conflicting		2				-10.121	Likely Pathogenic	0.523	Ambiguous	Likely Benign	0.228	Likely Benign	1.00	Ambiguous	0.0	0.27	Likely Benign	0.64	Ambiguous	1.02	Destabilizing	-0.86	Neutral	0.914	Possibly Damaging	0.921	Probably Damaging	3.23	Benign	0.03	Affected	3.37	32	3	4	-0.3	-14.03
c.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.1490A>G	Y497C (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.872	Likely Pathogenic	0.948	Likely Pathogenic	Ambiguous	0.806	Likely Pathogenic	3.88	Destabilizing	0.1	4.76	Destabilizing	4.32	Destabilizing	1.40	Destabilizing	-8.82	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.65	Pathogenic	0.03	Affected	3.37	35	0	-2	3.8	-60.04	209.9	59.1	-0.1	0.0	-0.3	0.1		X					X	Potentially Pathogenic	Tyr497 is located in the α-helix (res. Leu489-Glu519) within the inter-helix space of four α-helices (res. Leu489-Ile501, res. Val441-Ser457, res. Arg563-Glu578, res. Ala461-Val473). In the WT simulations, the phenol ring of Tyr497 hydrophobically packs with other residues in the inter-helix space (e.g., Leu465, Leu565, Val568). The hydroxyl group of Tyr497 also alternately forms hydrogen bonds with the carboxylate side chain of Gln456 and the backbone carbonyl of Glu564. Thus, Tyr497 plays a role in the folding and maintenance of the tertiary structure assembly between these four helices.In the variant simulations, the comparatively smaller residue, Cys497, cannot maintain any of the interactions seen with Tyr497 in the WT. Although no severe deleterious consequences are observed in the simulations, the structural effects could be more pronounced during actual protein folding. Indeed, the tertiary structure is seen to slightly break apart in the variant simulations.
c.1971G>C	W657C	Likely Pathogenic	GAP	Uncertain		1				-12.035	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.463	Likely Benign	2.74	Destabilizing	0.3	1.69	Ambiguous	2.22	Destabilizing	1.30	Destabilizing	-11.06	Deleterious	1.000	Probably Damaging	0.982	Probably Damaging	3.43	Benign	0.03	Affected			-8	-2	3.4	-83.07
c.2060G>A	R687Q (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1				-10.002	Likely Pathogenic	0.575	Likely Pathogenic	Likely Benign	0.401	Likely Benign	0.92	Ambiguous	0.1	-0.37	Likely Benign	0.28	Likely Benign	1.55	Destabilizing	-3.37	Deleterious	1.000	Probably Damaging	0.844	Possibly Damaging	3.91	Benign	0.03	Affected	3.42	17	1	1	1.0	-28.06
c.2095G>A	V699M (3D Viewer)		GAP	Uncertain		2	6-33441354-G-A	8	4.96e-6	-8.869	Likely Pathogenic	0.484	Ambiguous	Likely Benign	0.276	Likely Benign	-0.58	Ambiguous	0.1	0.29	Likely Benign	-0.15	Likely Benign	0.96	Ambiguous	-2.18	Neutral	0.994	Probably Damaging	0.806	Possibly Damaging	3.37	Benign	0.03	Affected	3.47	10	2	1	-2.3	32.06	257.8	-47.2	0.0	0.0	0.9	0.1						X		Potentially Benign	The isopropyl side chain of Val699, located on an α-helix (res. Leu685-Gln702), packs against hydrophobic residues (e.g., Leu703, Leu696, Leu435, Leu439) in the inter-helix space. In the variant simulations, the thioether side chain of Met699 has similar physicochemical properties to Val699 in the WT, and thus, it is able to maintain similar interactions. Consequently, the mutation causes no apparent changes in the structure.
c.2246G>A	R749Q	Likely Benign		Likely Benign		1	6-33441711-G-A	4	2.48e-6	-3.069	Likely Benign	0.212	Likely Benign	Likely Benign	0.152	Likely Benign										-1.00	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	2.64	Benign	0.03	Affected	4.32	2	1	1	1.0	-28.06
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.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.2444G>T	R815L	Likely Pathogenic	SH3-binding motif	Uncertain		1				-8.546	Likely Pathogenic	0.865	Likely Pathogenic	Ambiguous	0.175	Likely Benign										-3.06	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.63	Benign	0.03	Affected	4.32	4	-2	-3	8.3	-43.03
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.