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

c.dna	Variant	SGM Consensus	Domain	ClinVar			gnomAD			ESM1b		AlphaMissense			REVEL		FoldX			Rosetta		Foldetta		PremPS		PROVEAN		PolyPhen-2 HumDiv		PolyPhen-2 HumVar		FATHMM		SIFT				PAM		Physical		SASA		Normalized B-factor backbone		Normalized B-factor sidechain		SynGAP Structural Annotation									DOI
				Clinical Status	Review	Subm.	ID	Allele count	Allele freq.	LLR score	Prediction	Pathogenicity	Class	Optimized	Score	Prediction	Average ΔΔG	Prediction	StdDev	ΔΔG	Prediction	ΔΔG	Prediction	ΔΔG	Prediction	Score	Prediction	pph2_prob	Prediction	pph2_prob	Prediction	Nervous System Score	Prediction	Prediction	Status	Conservation	Sequences	PAM250	PAM120	Hydropathy Δ	MW Δ	Average	Δ	Δ	StdDev	Δ	StdDev	Secondary	Tertiary bonds	Inside out	GAP-Ras interface	At membrane	No effect	MD Alert	Verdict	Description
c.4021G>A	A1341T	Likely Benign		Conflicting		3	6-33451895-G-A	45	3.44e-5	-3.224	Likely Benign	0.081	Likely Benign	Likely Benign	0.099	Likely Benign										-0.58	Neutral	0.000	Benign	0.000	Benign	4.09	Benign	0.03	Affected	3.77	5	1	0	-2.5	30.03
c.4021G>T	A1341S	Likely Benign		Uncertain		1	6-33451895-G-T			-2.867	Likely Benign	0.078	Likely Benign	Likely Benign	0.099	Likely Benign										0.80	Neutral	0.000	Benign	0.001	Benign	4.40	Benign	1.00	Tolerated	3.77	5	1	1	-2.6	16.00
c.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.406C>T	R136W	Likely Pathogenic		Uncertain		2				-10.453	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.237	Likely Benign										-4.71	Deleterious	0.965	Probably Damaging	0.416	Benign	3.45	Benign	0.00	Affected	3.61	5	2	-3	3.6	30.03
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.407G>C	R136P	Likely Pathogenic		Uncertain		1				-11.952	Likely Pathogenic	0.981	Likely Pathogenic	Likely Pathogenic	0.277	Likely Benign										-3.72	Deleterious	0.910	Possibly Damaging	0.578	Possibly Damaging	3.47	Benign	0.00	Affected	3.61	5	0	-2	2.9	-59.07
c.416G>A	S139N	Likely Benign		Uncertain		1	6-33432713-G-A	3	2.22e-6	-4.584	Likely Benign	0.688	Likely Pathogenic	Likely Benign	0.109	Likely Benign										-0.75	Neutral	0.149	Benign	0.047	Benign	4.14	Benign	0.24	Tolerated	3.61	5	1	1	-2.7	27.03
c.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
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.453C>A	D151E	Likely Benign		Uncertain		1				-5.662	Likely Benign	0.886	Likely Pathogenic	Ambiguous	0.142	Likely Benign										-2.02	Neutral	0.984	Probably Damaging	0.967	Probably Damaging	3.99	Benign	0.11	Tolerated	3.61	5	3	2	0.0	14.03
c.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.458C>A	T153N	Likely Benign		Conflicting		3				-0.739	Likely Benign	0.226	Likely Benign	Likely Benign	0.161	Likely Benign										0.88	Neutral	0.888	Possibly Damaging	0.537	Possibly Damaging	4.23	Benign	0.81	Tolerated	3.61	5	0	0	-2.8	13.00
c.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.484C>G	R162G	Likely Benign		Uncertain		1				-6.985	Likely Benign	0.664	Likely Pathogenic	Likely Benign	0.190	Likely Benign										-0.73	Neutral	0.487	Possibly Damaging	0.272	Benign	4.09	Benign	0.78	Tolerated	3.74	4	-2	-3	4.1	-99.14
c.484C>T	R162C			Pathogenic		2				-8.157	Likely Pathogenic	0.787	Likely Pathogenic	Ambiguous	0.150	Likely Benign										-2.05	Neutral	0.988	Probably Damaging	0.513	Possibly Damaging	4.00	Benign	0.11	Tolerated	3.74	4	-4	-3	7.0	-53.05
c.485G>A	R162H			Uncertain		1	6-33432782-G-A	2	1.24e-6	-9.730	Likely Pathogenic	0.480	Ambiguous	Likely Benign	0.167	Likely Benign										-1.13	Neutral	0.957	Probably Damaging	0.513	Possibly Damaging	4.03	Benign	0.12	Tolerated	3.74	4	2	0	1.3	-19.05
c.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.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.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.515G>A	R172Q			Uncertain		1	6-33435157-G-A	3	1.86e-6	-7.245	In-Between	0.465	Ambiguous	Likely Benign	0.135	Likely Benign										-1.72	Neutral	0.804	Possibly Damaging	0.091	Benign	4.04	Benign	0.04	Affected	3.61	5	1	1	1.0	-28.06
c.526A>C	S176R	Likely Benign		Uncertain		1				-6.492	Likely Benign	0.987	Likely Pathogenic	Likely Pathogenic	0.247	Likely Benign										0.94	Neutral	0.718	Possibly Damaging	0.168	Benign	4.16	Benign	0.87	Tolerated			0	-1	-3.7	69.11
c.526A>G	S176G			Uncertain		1	6-33435168-A-G	1	6.20e-7	-7.541	In-Between	0.360	Ambiguous	Likely Benign	0.066	Likely Benign										-1.08	Neutral	0.131	Benign	0.039	Benign	4.08	Benign	0.22	Tolerated	3.54	6	0	1	0.4	-30.03
c.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.583G>C	A195P	Likely Pathogenic		Likely Pathogenic		1				-9.715	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.152	Likely Benign										-3.03	Deleterious	0.997	Probably Damaging	0.916	Probably Damaging	4.00	Benign	0.04	Affected	3.54	6	1	-1	-3.4	26.04
c.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.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.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.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.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.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.819G>T	E273D (3D Viewer)	Likely Benign	C2	Benign		1	6-33437724-G-T	2	1.24e-6	-1.811	Likely Benign	0.058	Likely Benign	Likely Benign	0.092	Likely Benign	0.26	Likely Benign	0.1	-0.48	Likely Benign	-0.11	Likely Benign	-0.63	Ambiguous	1.99	Neutral	0.004	Benign	0.010	Benign	2.00	Pathogenic	1.00	Tolerated	3.38	18	3	2	0.0	-14.03	223.1	22.1	0.2	0.0	0.0	0.1						X		Potentially Benign	The negatively charged residue Glu273, located in a β hairpin loop (res. Glu273-Lys278) that connects two anti-parallel β sheet strands, is replaced with another negatively charged residue, aspartate. Because the C2 domain loop faces the membrane surface, the potentially crucial role of the carboxylate group of Glu273 or Asp273 on SynGAP-membrane association cannot be fully explored via solvent-only simulations.As a minor note, the neighboring residue Arg272, which stacks with the indole ring of the Trp362 side chain and directly faces RasGTPase, forms a salt bridge more often with Asp273 than with the non-mutated Glu273 in the simulations. Regardless, due to the similar physicochemical properties of the WT and variant residues at the membrane interface, the residue swap is likely to be well tolerated.
c.1663G>A	V555I (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.544	Likely Benign	0.084	Likely Benign	Likely Benign	0.253	Likely Benign	-0.82	Ambiguous	0.0	-0.41	Likely Benign	-0.62	Ambiguous	-0.55	Ambiguous	0.45	Neutral	0.002	Benign	0.002	Benign	-1.26	Pathogenic	1.00	Tolerated			4	3	0.3	14.03
c.1136C>T	S379L (3D Viewer)	Likely Benign	C2	Benign		1	6-33438041-C-T	8	4.05e-5	-5.641	Likely Benign	0.173	Likely Benign	Likely Benign	0.469	Likely Benign	0.39	Likely Benign	0.2	3.38	Destabilizing	1.89	Ambiguous	-0.52	Ambiguous	-0.85	Neutral	0.015	Benign	0.002	Benign	3.83	Benign	0.04	Affected	4.32	11	-3	-2	4.6	26.08	251.9	-48.1	0.6	1.1	0.0	0.5								Uncertain	Ser379 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like leucine are rarely tolerated. Although no negative structural effects are observed in the variant simulations, Leu379 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1027G>A	V343I (3D Viewer)	Likely Benign	C2	Uncertain		2	6-33437932-G-A	1	6.20e-7	-6.020	Likely Benign	0.117	Likely Benign	Likely Benign	0.020	Likely Benign	-0.27	Likely Benign	0.0	-0.04	Likely Benign	-0.16	Likely Benign	-0.39	Likely Benign	-0.14	Neutral	0.159	Benign	0.084	Benign	1.98	Pathogenic	0.27	Tolerated	3.37	25	4	3	0.3	14.03	240.2	-26.9	-0.2	0.2	-0.2	0.2						X		Potentially Benign	The iso-propyl side chain of Val343, located in an anti-parallel β sheet strand (res. Gly341-Pro349), is packing against multiple hydrophobic residues of the C2 domain (e.g., Leu327, Leu274, Val365). In the variant simulations, the sec-butyl side chain of Ile343 is basically able to form the same interactions as valine due to its similar hydrophobic profile. The residue swap also does not seem to cause negative effects on the protein structure based on the simulations.
c.2101C>T	P701S (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441360-C-T	3	1.86e-6	-4.375	Likely Benign	0.221	Likely Benign	Likely Benign	0.132	Likely Benign	1.33	Ambiguous	0.0	0.12	Likely Benign	0.73	Ambiguous	-0.36	Likely Benign	0.78	Neutral	0.044	Benign	0.025	Benign	3.48	Benign	1.00	Tolerated	3.47	10	-1	1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.2186A>G	N729S (3D Viewer)	Likely Benign	GAP	Uncertain		1				-1.578	Likely Benign	0.066	Likely Benign	Likely Benign	0.063	Likely Benign	0.14	Likely Benign	0.1	1.34	Ambiguous	0.74	Ambiguous	-0.36	Likely Benign	-0.42	Neutral	0.221	Benign	0.027	Benign	3.38	Benign	0.93	Tolerated	3.59	7	1	1	2.7	-27.03
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.1142G>T	G381V (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438047-G-T	2	1.25e-6	-5.967	Likely Benign	0.146	Likely Benign	Likely Benign	0.618	Likely Pathogenic	7.16	Destabilizing	1.0	4.10	Destabilizing	5.63	Destabilizing	-0.32	Likely Benign	-0.95	Neutral	0.386	Benign	0.157	Benign	1.32	Pathogenic	0.10	Tolerated	4.32	9	-1	-3	4.6	42.08	214.6	-68.8	0.3	0.7	-0.5	0.3								Uncertain	Gly381 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like valine are rarely tolerated. Although no negative structural effects are observed in the variant simulations, Val381 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effects on Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1811C>T	S604L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440863-C-T	6	3.72e-6	-14.683	Likely Pathogenic	0.965	Likely Pathogenic	Likely Pathogenic	0.639	Likely Pathogenic	-0.94	Ambiguous	0.1	-1.24	Ambiguous	-1.09	Ambiguous	-0.31	Likely Benign	-5.97	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	3.09	Benign	0.00	Affected	3.37	35	-3	-2	4.6	26.08	234.0	-49.6	0.0	0.1	0.3	0.5	X			X				Potentially Pathogenic	Ser604 is located in a short turn between an α helix (res. Glu582-Met603) and a short α helical section (res. Ser606-Phe608). In the WT simulations, the hydroxyl side chain of Ser604 periodically hydrogen bonds with the backbone carbonyl groups of other α helix residues (e.g., Pro600, Met603). Serine weakens the α helix secondary structure, and thus, Ser604 along with Pro605 breaks the α helix, facilitating the turn in the WT structure.In contrast, in the variant simulations, Leu604 forms a few hydrophobic interactions (e.g., Leu607, Phe608). More importantly, the helix end is more stable than with Ser604 in the WT. The residue swap could have a more profound effect on the actual folding process, for example, by preventing the bending at the α helix end, than what the simulations suggest.Moreover, Ser604 directly hydrogen bonds with Ras residues Ser65 and Ala66 in the WT SynGAP-Ras complex. The hydrophobic leucine cannot maintain these interactions with Ras at the GAP-Ras interface. Thus, the effect of the residue swap on the complex formation with the GTPase cannot be fully explored in the solvent-only simulations.
c.1918A>T	T640S (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441177-A-T	1	6.20e-7	-2.371	Likely Benign	0.062	Likely Benign	Likely Benign	0.088	Likely Benign	-0.78	Ambiguous	0.1	0.43	Likely Benign	-0.18	Likely Benign	-0.30	Likely Benign	0.92	Neutral	0.000	Benign	0.001	Benign	3.60	Benign	0.33	Tolerated	3.37	30	1	1	-0.1	-14.03
c.1198G>C	V400L (3D Viewer)	Likely Benign	C2	Benign		1	6-33438103-G-C	22	1.36e-5	-1.000	Likely Benign	0.137	Likely Benign	Likely Benign	0.325	Likely Benign	-0.71	Ambiguous	0.2	0.39	Likely Benign	-0.16	Likely Benign	-0.29	Likely Benign	-0.60	Neutral	0.001	Benign	0.001	Benign	5.33	Benign	0.64	Tolerated	3.38	27	2	1	-0.4	14.03	251.0	-30.1	0.0	0.0	0.7	0.1						X		Potentially Benign	The iso-propyl side chain of Val400, located in an anti-parallel β sheet strand (res. Ala399-Ile411), hydrophobically packs against hydrophobic residues within the anti-parallel β sheet of the C2 domain (e.g., Ile268, Ala404, Leu325, Leu402). Val400 is swapped for another hydrophobic residue, leucine, whose branched hydrocarbon side chain is of a comparable size and thus packs favorably within the C2 domain. In short, the residue swap has no apparent negative effect on the structure based on the variant simulations.	10.1016/j.ajhg.2020.11.011
c.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.1964T>A	L655Q (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.278	Likely Benign	0.144	Likely Benign	Likely Benign	0.139	Likely Benign	-0.01	Likely Benign	0.0	0.69	Ambiguous	0.34	Likely Benign	-0.15	Likely Benign	0.61	Neutral	0.955	Possibly Damaging	0.602	Possibly Damaging	3.59	Benign	0.65	Tolerated	3.39	24	-2	-2	-7.3	14.97	229.9	-8.6	0.0	0.0	0.4	0.0						X		Potentially Benign	The iso-butyl side chain of Leu655, located on the surface of an α helix (res. Ser641-Glu666), is not involved in any interactions in the WT simulations. In the variant simulations, the carboxamide side chain of Gln655 dynamically interacts with neighboring residues (e.g., Glu651, Glu656, Arg544) on the protein surface, with no negative structural effects.
c.1579G>T	D527Y (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.386	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.905	Likely Pathogenic	-0.77	Ambiguous	0.2	1.89	Ambiguous	0.56	Ambiguous	-0.14	Likely Benign	-8.79	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-2.41	Pathogenic	0.00	Affected	3.37	35	-4	-3	2.2	48.09	270.9	-45.7	0.1	0.1	-0.1	0.0		X						Potentially Pathogenic	Asp527 is located on an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the carboxylate group of the Asp527 side chain forms hydrogen bonds with the backbone atoms of loop residues (e.g., Ile529, Lys530) facing the membrane surface. In the variant simulations, Tyr527 is a bulkier residue that faces away from the loop and stacks with Phe646 in a nearby α-helix (res. Ser614-Ser668). Regardless, no negative structural effects are observed during the variant simulations. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.
c.1685C>T	P562L (3D Viewer)	Likely Pathogenic	GAP	Pathogenic/Likely path.		10	6-33440737-C-T			-13.438	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.829	Likely Pathogenic	3.54	Destabilizing	0.8	0.17	Likely Benign	1.86	Ambiguous	-0.14	Likely Benign	-9.95	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	0.58	Pathogenic	0.00	Affected	3.37	35	-3	-3	5.4	16.04	228.8	-68.5	-0.1	0.0	0.1	0.2							X	Potentially Pathogenic	Pro562 is located on an α-α loop between two α-helices (res. Ala533-Val560 and res. Arg563-Glu578). The cyclic pyrrolidine side chain of Pro562 hydrophobically packs with other residues in the inter-helix space, such as Leu565, Ile501, and Phe561. In the variant simulations, Leu562 packs more favorably with the nearby hydrophobic residues, and the backbone amide group of Leu562 (absent in proline) does not form any intra-protein hydrogen bonds. However, prolines are well-suited for unstructured regions like loops, and thus, Pro562 in the WT is necessary at the end of the helix to induce a tight turn during folding. Although no negative structural effects are observed during the simulations, the residue swap could potentially cause extensive damage to the protein structure during folding.	10.1016/j.ajhg.2020.11.011
c.1172G>T	G391V (3D Viewer)	Likely Benign	C2	Likely Benign		1	6-33438077-G-T	3	1.86e-6	-6.642	Likely Benign	0.133	Likely Benign	Likely Benign	0.595	Likely Pathogenic	4.23	Destabilizing	1.3	4.81	Destabilizing	4.52	Destabilizing	-0.11	Likely Benign	-0.98	Neutral	0.994	Probably Damaging	0.887	Possibly Damaging	1.32	Pathogenic	0.10	Tolerated	3.69	8	-1	-3	4.6	42.08	228.6	-69.0	0.0	0.8	-0.5	0.3								Uncertain	Gly387 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364 and res. Ala399-Ile411). The Ω loop is assumed to directly interact with the membrane, and it is observed to move arbitrarily throughout the WT solvent simulations. This loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play significant roles in protein functions that require flexibility, and thus hydrophobic residues like valine are rarely tolerated. Although no negative structural effects are visualized in the variant’s simulations, Val391 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. Since the effects on the Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1040C>A	T347N (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33437945-C-A	9	5.58e-6	-5.545	Likely Benign	0.165	Likely Benign	Likely Benign	0.059	Likely Benign	0.41	Likely Benign	0.1	0.46	Likely Benign	0.44	Likely Benign	-0.06	Likely Benign	1.96	Neutral	0.001	Benign	0.001	Benign	1.67	Pathogenic	0.60	Tolerated	3.37	25	0	0	-2.8	13.00
c.1417G>A	V473I (3D Viewer)		GAP	Uncertain		1	6-33438449-G-A	1	6.20e-7	-7.481	In-Between	0.418	Ambiguous	Likely Benign	0.203	Likely Benign	-0.12	Likely Benign	0.0	1.20	Ambiguous	0.54	Ambiguous	-0.06	Likely Benign	-0.91	Neutral	0.929	Possibly Damaging	0.917	Probably Damaging	3.74	Benign	0.18	Tolerated	3.37	34	3	4	0.3	14.03
c.1786C>T	R596C (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2	6-33440838-C-T	6	3.72e-6	-10.805	Likely Pathogenic	0.972	Likely Pathogenic	Likely Pathogenic	0.633	Likely Pathogenic	2.94	Destabilizing	0.0	1.49	Ambiguous	2.22	Destabilizing	-0.03	Likely Benign	-7.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.41	Pathogenic	0.00	Affected	3.37	35	-4	-3	7.0	-53.05	230.7	97.9	-0.1	0.0	-0.3	0.4		X		X				Potentially Pathogenic	The guanidinium group of Arg596, located in an α helix (res. Glu582-Met603), forms a salt bridge with the carboxylate group of Glu495 from another α helix (res. Leu489-Glu519). In the WT simulations, the side chain of Arg596 hydrogen bonds with the backbone carbonyl groups of Asn487, Glu486, Arg485, and Phe484. Additionally, Arg596 can hydrogen bond with the carboxamide group of the Asn487 side chain on an opposing loop that links two α helices (res. Ala461-Arg475, res. Leu489-Glu519).In the variant simulations, the thiol group of the Cys596 side chain is unable to form salt bridges or any of the hydrogen bonds that the Arg596 side chain can. Thus, the residue swap could affect the tertiary structure assembly more profoundly than observed in the simulations. Notably, Arg596 plays a key role in positioning the aforementioned loop, which is crucial for the placement of the “arginine finger” or the Arg485 side chain during RasGTPase activation.
c.862G>A	D288N (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437767-G-A	2	1.24e-6	-10.535	Likely Pathogenic	0.521	Ambiguous	Likely Benign	0.321	Likely Benign	-0.39	Likely Benign	0.1	0.01	Likely Benign	-0.19	Likely Benign	-0.03	Likely Benign	-3.73	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.78	Pathogenic	0.05	Affected	3.38	23	1	2	0.0	-0.98
c.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.1108G>A	G370S (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438013-G-A	15	9.31e-6	-3.533	Likely Benign	0.081	Likely Benign	Likely Benign	0.282	Likely Benign	2.83	Destabilizing	2.0	1.05	Ambiguous	1.94	Ambiguous	-0.02	Likely Benign	0.47	Neutral	0.000	Benign	0.000	Benign	1.33	Pathogenic	0.77	Tolerated	3.42	19	1	0	-0.4	30.03	196.6	-49.6	0.9	2.2	-0.1	0.4								Uncertain	Gly370 is located in the Gly-rich Ω loop (res. Pro364- Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because, the Ω loop is assumed to be directly interacting with the membrane, it is only seen to move arbitrarily throughout the WT solvent simulations. The Ω loop is potentially playing a crucial loop in the SynGAP-membrane complex association, stability and dynamics, regardless, this aspect cannot be addressed through the solvent simulations only. The Ω-loops are known to have a major role in protein functions that requires flexibility and thus, they are rich in glycines, prolines and to a lesser extent, hydrophilic residues to ensure maximum flexibility. Thus, Ser370 in the variant is potentially tolerated in the Ω loop. However, since the effect on the Gly-rich Ω loop dynamics can only be well-studied through the SynGAP-membrane complex, no definite conclusions can be withdrawn.
c.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.2113A>C	K705Q (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441372-A-C	1	6.20e-7	-5.787	Likely Benign	0.436	Ambiguous	Likely Benign	0.142	Likely Benign	-0.10	Likely Benign	0.0	0.33	Likely Benign	0.12	Likely Benign	-0.02	Likely Benign	-0.24	Neutral	0.997	Probably Damaging	0.969	Probably Damaging	3.42	Benign	0.78	Tolerated	3.47	10	1	1	0.4	-0.04
c.1787G>T	R596L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.197	Likely Pathogenic	0.992	Likely Pathogenic	Likely Pathogenic	0.756	Likely Pathogenic	1.51	Ambiguous	0.3	-0.58	Ambiguous	0.47	Likely Benign	-0.02	Likely Benign	-6.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.45	Pathogenic	0.00	Affected	3.37	35	-3	-2	8.3	-43.03	234.2	63.4	-0.1	0.0	-0.5	0.6		X		X				Potentially Pathogenic	The guanidinium group of Arg596, located in an α helix (res. Glu582-Met603), forms a salt bridge with the carboxylate group of Glu495 from another α helix (res. Leu489-Glu519). In the WT simulations, the side chain of Arg596 hydrogen bonds with the backbone carbonyl groups of Asn487, Glu486, Arg485, and Phe484. Additionally, Arg596 can hydrogen bond with the carboxamide group of the Asn487 side chain on an opposing loop that links two α helices (res. Ala461-Arg475, res. Leu489-Glu519).However, in the variant simulations, the branched hydrocarbon side chain of Leu596 cannot form any of the hydrogen bonds or salt bridges maintained by the considerably bulkier and positively charged Arg596 side chain. Instead, Leu596 packs hydrophobically with the phenyl ring of Phe484 in the linker loop or residues from the opposing helix (e.g., Ile494, Thr491).Thus, the residue swap could affect the tertiary structure assembly more profoundly than observed in the simulations. Notably, Arg596 plays a key role in positioning the aforementioned loop, which is crucial for the placement of the “arginine finger” or the Arg485 side chain during RasGTPase activation.	10.1016/j.ajhg.2020.11.011
c.866T>C	M289T	Likely Benign	C2	Uncertain		1				-4.668	Likely Benign	0.238	Likely Benign	Likely Benign	0.222	Likely Benign	0.73	Ambiguous	0.1	0.17	Likely Benign	0.45	Likely Benign	-0.01	Likely Benign	-0.47	Neutral	0.801	Possibly Damaging	0.315	Benign	1.83	Pathogenic	0.57	Tolerated			-1	-1	-2.6	-30.09
c.1154C>G	S385W (3D Viewer)		C2	Benign		1	6-33438059-C-G			-9.353	Likely Pathogenic	0.362	Ambiguous	Likely Benign	0.373	Likely Benign	0.53	Ambiguous	0.2	0.69	Ambiguous	0.61	Ambiguous	0.00	Likely Benign	-0.84	Neutral	0.986	Probably Damaging	0.968	Probably Damaging	4.63	Benign	0.00	Affected	4.32	3	-2	-3	-0.1	99.14	260.4	-71.2	0.5	1.3	0.7	0.4								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like tryptophan are rarely tolerated. Although no major negative structural effects are observed in the variant simulations, Trp385 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effects on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.	10.1016/j.ajhg.2020.11.011
c.1556A>C	E519A (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-8.557	Likely Pathogenic	0.904	Likely Pathogenic	Ambiguous	0.384	Likely Benign	-0.05	Likely Benign	0.0	0.55	Ambiguous	0.25	Likely Benign	0.00	Likely Benign	-5.23	Deleterious	0.999	Probably Damaging	0.998	Probably Damaging	3.33	Benign	0.10	Tolerated	3.37	35	0	-1	5.3	-58.04	162.4	83.5	-0.1	0.1	-0.2	0.0						X		Potentially Benign	Glu519 is located at the beginning of an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the carboxylate side chain of Glu519 does not make any specific interactions. Accordingly, the Ala residue swap does not show any negative structural effects in the variant simulations. However, it should be noted that Glu519 faces the missing part of the N-terminal in the model, and thus its potential role in maintaining the tertiary structure might be de-emphasized in the current model.
c.2003C>T	S668F (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-15.047	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.643	Likely Pathogenic	16.72	Destabilizing	5.0	11.07	Destabilizing	13.90	Destabilizing	0.00	Likely Benign	-5.98	Deleterious	0.999	Probably Damaging	0.935	Probably Damaging	3.18	Benign	0.00	Affected	3.38	28	-3	-2	3.6	60.10	250.9	-59.6	-0.1	0.1	0.0	0.1		X	X				X	Potentially Pathogenic	In the WT simulations, the hydroxyl side chain of Ser668, located on an α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), forms hydrogen bonds with the backbone carbonyl groups of Leu664, Tyr665, and Glu666, as well as the guanidinium group of Arg573 on a nearby α-helix (res. Arg563-Glu578). In the variant simulations, the side chain of Phe668 cannot maintain the same hydrogen-bond network. Due to its larger size, it moves away to avoid steric hindrance. In the WT simulations, a network of hydrogen bonds between several residues (e.g., Asn669, Lys566, and Glu666) keeps both α-helices and the proceeding loop (res. Asn669-Asp684) tightly connected, but this setup is not present in the variant simulations. Additionally, in the variant simulations, the side chain of Arg573 shifts to form a more stable salt bridge with the carboxylate group of Glu582 instead of hydrogen bonding with Ser668 as in the WT simulations.
c.958G>A	V320I (3D Viewer)	Likely Benign	C2	Uncertain		1				-5.220	Likely Benign	0.111	Likely Benign	Likely Benign	0.027	Likely Benign	-0.27	Likely Benign	0.2	0.66	Ambiguous	0.20	Likely Benign	0.01	Likely Benign	-0.21	Neutral	0.198	Benign	0.114	Benign	1.77	Pathogenic	0.45	Tolerated	3.38	23	3	4	0.3	14.03
c.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.1339G>C	V447L (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.136	Likely Benign	0.491	Ambiguous	Likely Benign	0.180	Likely Benign	-1.13	Ambiguous	0.1	0.54	Ambiguous	-0.30	Likely Benign	0.03	Likely Benign	-0.29	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.61	Benign	0.90	Tolerated	3.37	32	1	2	-0.4	14.03
c.901G>A	A301T (3D Viewer)	Likely Benign	C2	Uncertain		5	6-33437806-G-A	2	1.24e-6	-3.448	Likely Benign	0.070	Likely Benign	Likely Benign	0.150	Likely Benign	0.36	Likely Benign	0.2	-0.33	Likely Benign	0.02	Likely Benign	0.03	Likely Benign	-0.25	Neutral	0.997	Probably Damaging	0.989	Probably Damaging	4.15	Benign	0.22	Tolerated	4.32	14	1	0	-2.5	30.03	219.8	-42.8	-0.1	0.0	-0.5	0.2								Uncertain	The methyl group of Ala301, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Met289-Pro298, res. Thr305-Asn315), points outward from the β hairpin loop, and its backbone atoms do not participate in the loop formation in the WT simulations. In the variant simulations, the hydroxyl group of the Thr301 side chain also mostly points outward; however, the guanidinium group of 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.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.1851G>T	E617D (3D Viewer)	Likely Benign	GAP	Uncertain		1				-1.349	Likely Benign	0.241	Likely Benign	Likely Benign	0.322	Likely Benign	0.12	Likely Benign	0.1	0.80	Ambiguous	0.46	Likely Benign	0.07	Likely Benign	-0.01	Neutral	0.994	Probably Damaging	0.979	Probably Damaging	-1.35	Pathogenic	0.88	Tolerated	3.37	35	2	3	0.0	-14.03
c.1147G>T	G383W (3D Viewer)		C2	Uncertain		1	6-33438052-G-T	1	6.22e-7	-10.161	Likely Pathogenic	0.439	Ambiguous	Likely Benign	0.469	Likely Benign	5.81	Destabilizing	3.6	4.44	Destabilizing	5.13	Destabilizing	0.08	Likely Benign	-1.01	Neutral	0.959	Probably Damaging	0.704	Possibly Damaging	4.09	Benign	0.00	Affected	4.32	7	-2	-7	-0.5	129.16
c.1594A>C	T532P (3D Viewer)	Likely Benign	GAP	Benign		1				-2.143	Likely Benign	0.061	Likely Benign	Likely Benign	0.201	Likely Benign	-0.30	Likely Benign	0.2	0.06	Likely Benign	-0.12	Likely Benign	0.08	Likely Benign	-0.90	Neutral	0.005	Benign	0.008	Benign	-1.28	Pathogenic	0.18	Tolerated	3.37	35	0	-1	-0.9	-3.99	174.2	35.1	0.4	0.0	0.1	0.0						X		Potentially Benign	Thr532 is located on an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560) facing the membrane. In the WT simulations, the hydroxyl group of Thr532 occasionally forms hydrogen bonds with the backbone atoms of other loop residues without any specific interaction. In the variant simulations, the Pro532 residue swap does not cause structural changes. Although hydrophilic residues seem more favorable in the loop, the pyrrolidine side chain of proline is well suited for unstructured protein regions such as loops. However, due to its location at the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.
c.2116G>A	E706K (3D Viewer)		GAP	Uncertain		1				-10.519	Likely Pathogenic	0.833	Likely Pathogenic	Ambiguous	0.080	Likely Benign	1.17	Ambiguous	0.1	0.51	Ambiguous	0.84	Ambiguous	0.08	Likely Benign	-1.51	Neutral	0.345	Benign	0.028	Benign	4.15	Benign	0.52	Tolerated	3.47	10	0	1	-0.4	-0.94	187.1	49.2	0.0	0.0	0.4	0.1						X		Uncertain	The carboxylate side chain of Glu706, located at the end and outer surface of an α-helix (res. Thr704-Gly712), forms a salt bridge with Lys710 and a hydrogen bond with its own backbone amino group at the helix end in the WT simulations. Although Lys706 is unable to make these transient interactions in the variant simulations, there is no apparent negative effect on the protein structure due to the residue swap. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.1118G>T	G373V (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438023-G-T	6	5.03e-6	-6.062	Likely Benign	0.112	Likely Benign	Likely Benign	0.428	Likely Benign	5.32	Destabilizing	3.2	0.82	Ambiguous	3.07	Destabilizing	0.09	Likely Benign	-0.98	Neutral	0.007	Benign	0.001	Benign	3.90	Benign	0.00	Affected	3.53	16	-1	-3	4.6	42.08	207.6	-68.1	1.9	1.1	-0.6	0.1								Uncertain	Gly373 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like valine are rarely tolerated. Although no negative structural effects are observed in the variant simulations, Val373 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on the Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.611C>G	S204C (3D Viewer)	Likely Benign	PH	Uncertain		1				-6.613	Likely Benign	0.127	Likely Benign	Likely Benign	0.148	Likely Benign	0.65	Ambiguous	0.4	-1.13	Ambiguous	-0.24	Likely Benign	0.10	Likely Benign	-0.64	Neutral	0.978	Probably Damaging	0.753	Possibly Damaging	4.13	Benign	0.05	Affected	3.44	10	0	-1	3.3	16.06	223.6	-13.8	0.6	0.3	0.0	0.2	X							Uncertain	The hydroxyl-containing Ser204, located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by the thiol-containing cysteine. In the WT simulations, Ser204 simultaneously forms hydrogen bonds with the backbone carbonyl of Asp201 and the hydroxyl group of Thr224, helping to stabilize the two anti-parallel β strands (res. Ile205-Lys207 and Cys219-Thr223) at the end of the β sheet. Since the thiol group of cysteine forms weaker hydrogen bonds than the hydroxyl group of serine, Cys204 does not maintain the hydrogen bond network as stably as Ser204 in the variant simulations. However, because the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.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.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.2029A>T	S677C (3D Viewer)	Likely Benign	GAP	Benign		1				-8.496	Likely Pathogenic	0.076	Likely Benign	Likely Benign	0.153	Likely Benign	-0.51	Ambiguous	0.3	-0.30	Likely Benign	-0.41	Likely Benign	0.15	Likely Benign	-2.41	Neutral	0.932	Possibly Damaging	0.222	Benign	3.25	Benign	0.04	Affected	3.41	23	-1	0	3.3	16.06
c.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.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.1667A>G	N556S (3D Viewer)		GAP	Uncertain		1	6-33438910-A-G	3	1.86e-6	-6.576	Likely Benign	0.197	Likely Benign	Likely Benign	0.449	Likely Benign	0.52	Ambiguous	0.1	0.14	Likely Benign	0.33	Likely Benign	0.16	Likely Benign	-3.60	Deleterious	1.000	Probably Damaging	0.989	Probably Damaging	-1.22	Pathogenic	0.14	Tolerated	3.37	35	1	1	2.7	-27.03	198.8	31.0	0.0	0.0	-0.5	0.2						X		Potentially Benign	Asn556 is located on the outer surface of an α-helix (res. Ala533-Val560). The carboxamide group of Asn556 forms hydrogen bonds with nearby residues such as Lys553 and Cys552. It also forms a hydrogen bond with the backbone carbonyl group of Cys552, which weakens the α-helix integrity. In the variant simulations, the hydroxyl group of Ser556 forms a more stable hydrogen bond with the backbone carbonyl oxygen of the same helix residue, Cys552, compared to Asn556 in the WT. Serine has a slightly lower propensity to reside in an α-helix than asparagine, which may exacerbate the negative effect on the α-helix integrity. However, the residue swap does not cause negative structural effects during the simulations.
c.1586T>C	I529T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-0.539	Likely Benign	0.336	Likely Benign	Likely Benign	0.343	Likely Benign	0.22	Likely Benign	0.2	0.16	Likely Benign	0.19	Likely Benign	0.17	Likely Benign	0.24	Neutral	0.872	Possibly Damaging	0.820	Possibly Damaging	-1.23	Pathogenic	0.55	Tolerated	3.37	35	0	-1	-5.2	-12.05	207.2	29.8	0.2	0.0	0.2	0.1						X		Potentially Benign	Ile529 is located on an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the sec-butyl side chain of Ile529 faces the membrane interface and shows no specific interactions. In the variant simulations, the hydroxyl group of Thr529 forms a hydrogen bond with the carboxylate side chain of Asp527, but no negative structural changes are observed. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.
c.1604G>C	S535T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33438847-G-C	14	8.67e-6	-3.886	Likely Benign	0.069	Likely Benign	Likely Benign	0.177	Likely Benign	0.45	Likely Benign	0.1	-0.27	Likely Benign	0.09	Likely Benign	0.17	Likely Benign	-0.81	Neutral	0.000	Benign	0.001	Benign	-1.25	Pathogenic	0.25	Tolerated	3.37	35	1	1	0.1	14.03	201.3	-17.3	-0.1	0.7	-0.2	0.1						X		Potentially Benign	Ser535 is located near the terminal end of an α-helix (res. Ala533-Val560) close to the membrane interface. In the WT simulations, the hydroxyl side chain of Ser535 forms hydrogen bonds with nearby residues (e.g., His539, Glu538) without any specific interactions. These hydrogen bonds disrupt the structure of the terminal end of the α-helix (Ala533-Ser535), causing it to weaken or unfold during the WT simulations. In the variant simulations, Thr535, a hydrophilic residue with a hydroxyl group of almost the same size as Ser, interacts more frequently with the preceding loop residues (e.g., Thr532, Cys531) due to its longer side chain. Regardless, the residue swap is tolerated in the simulations with no negative effects. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.	10.1016/j.ajhg.2020.11.011
c.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.1150G>A	G384S (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438055-G-A	1	6.22e-7	-5.243	Likely Benign	0.090	Likely Benign	Likely Benign	0.315	Likely Benign	1.92	Ambiguous	0.2	1.66	Ambiguous	1.79	Ambiguous	0.19	Likely Benign	-0.67	Neutral	0.980	Probably Damaging	0.968	Probably Damaging	1.33	Pathogenic	0.04	Affected	4.32	2	1	0	-0.4	30.03	202.4	-49.8	0.5	1.0	-0.2	0.0								Uncertain	Gly384 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycines, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Ser384 is potentially tolerated in the Ω loop, although the hydroxyl group of Ser384 forms various hydrogen bonds with several other loop residues in the variant simulations. However, since the effects on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1153T>C	S385P (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438058-T-C			-5.431	Likely Benign	0.123	Likely Benign	Likely Benign	0.385	Likely Benign	0.91	Ambiguous	0.6	-0.90	Ambiguous	0.01	Likely Benign	0.19	Likely Benign	-0.26	Neutral	0.676	Possibly Damaging	0.693	Possibly Damaging	4.63	Benign	0.04	Affected	4.32	3	1	-1	-0.8	10.04	210.3	18.5	1.8	0.9	0.3	0.0								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycine residues, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Pro385 is potentially tolerated in the Ω loop. However, since the effects on Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1441C>T	H481Y (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438473-C-T	16	9.91e-6	-10.910	Likely Pathogenic	0.565	Likely Pathogenic	Likely Benign	0.256	Likely Benign	-0.53	Ambiguous	0.1	-0.46	Likely Benign	-0.50	Ambiguous	0.20	Likely Benign	-3.32	Deleterious	0.988	Probably Damaging	0.979	Probably Damaging	3.40	Benign	0.59	Tolerated	3.37	33	0	2	1.9	26.03	256.5	-44.4	0.0	0.0	0.2	0.2	X			X				Uncertain	The imidazole ring of the His481 side chain is located in a short helical structure (res. Glu480-Leu482) within an α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. In the WT simulations, His481 alternately stacks against Arg485, Arg587, and Glu480 without a definite role. In the variant simulations, Tyr481 also alternately stacks with nearby arginine residues, including Arg485, Arg587, and Arg479. The interaction between Tyr481 and Arg479 affects the α-α loop, causing it to fold into a distorted helical structure, an effect that might be more pronounced during protein folding. Finally, the potential effect of the residue swap on SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.910G>A	D304N (3D Viewer)		C2	Uncertain		1				-6.194	Likely Benign	0.391	Ambiguous	Likely Benign	0.345	Likely Benign	0.30	Likely Benign	0.1	-0.08	Likely Benign	0.11	Likely Benign	0.21	Likely Benign	-4.18	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.81	Pathogenic	0.03	Affected	3.38	23	1	2	0.0	-0.98
c.603T>A	D201E (3D Viewer)	Likely Benign	PH	Benign		1				-2.640	Likely Benign	0.406	Ambiguous	Likely Benign	0.165	Likely Benign	0.42	Likely Benign	0.2	1.99	Ambiguous	1.21	Ambiguous	0.23	Likely Benign	-0.69	Neutral	0.633	Possibly Damaging	0.108	Benign	4.30	Benign	1.00	Tolerated	3.46	9	3	2	0.0	14.03	258.7	-24.8	0.9	0.1	-0.3	0.2						X		Uncertain	Asp201, an acidic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another acidic residue, glutamate. The carboxylate groups of both Asp201 and Glu201 side chains form hydrogen bonds with the hydroxyl group of Ser221 in the simulations. Due to its shorter side chain, Asp201 can also hydrogen bond with the backbone amide groups of neighboring loop residues Ser204 and Asp203. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.603T>G	D201E (3D Viewer)	Likely Benign	PH	Conflicting		2	6-33435245-T-G	20	1.24e-5	-2.640	Likely Benign	0.406	Ambiguous	Likely Benign	0.165	Likely Benign	0.42	Likely Benign	0.2	1.99	Ambiguous	1.21	Ambiguous	0.23	Likely Benign	-0.69	Neutral	0.633	Possibly Damaging	0.108	Benign	4.30	Benign	1.00	Tolerated	3.46	9	3	2	0.0	14.03	258.7	-24.8	0.9	0.1	-0.3	0.2						X		Uncertain	Asp201, an acidic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another acidic residue, glutamate. The carboxylate groups of both Asp201 and Glu201 side chains form hydrogen bonds with the hydroxyl group of Ser221 in the simulations. Due to its shorter side chain, Asp201 can also hydrogen bond with the backbone amide groups of neighboring loop residues Ser204 and Asp203. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.1131G>A	M377I (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438036-G-A	1	6.23e-7	-2.895	Likely Benign	0.212	Likely Benign	Likely Benign	0.227	Likely Benign	0.76	Ambiguous	0.3	0.54	Ambiguous	0.65	Ambiguous	0.24	Likely Benign	-0.41	Neutral	0.000	Benign	0.001	Benign	5.46	Benign	0.26	Tolerated	4.32	12	1	2	2.6	-18.03
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.865A>G	M289V (3D Viewer)	Likely Benign	C2	Benign		1				-4.239	Likely Benign	0.117	Likely Benign	Likely Benign	0.150	Likely Benign	1.09	Ambiguous	0.1	-0.27	Likely Benign	0.41	Likely Benign	0.24	Likely Benign	-0.36	Neutral	0.136	Benign	0.054	Benign	1.80	Pathogenic	1.00	Tolerated	3.38	23	2	1	2.3	-32.06	204.2	51.0	0.0	0.0	0.2	0.0						X		Potentially Benign	The hydrophobic residue Met289, located in a β hairpin linking two anti-parallel β sheet strands (res. Met289-Arg299, res. Arg272-Leu286), is swapped for another hydrophobic residue, valine. In the variant simulations, the branched hydrocarbon side chain of Val289 packs against the phenol group of the Tyr291 side chain but is unable to form methionine-aromatic interactions. β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant. However, based on the simulations, the residue swap does not cause adverse effects on the structure.
c.1354G>A	V452I (3D Viewer)		GAP	Uncertain		1				-8.985	Likely Pathogenic	0.361	Ambiguous	Likely Benign	0.218	Likely Benign	-0.08	Likely Benign	0.1	0.51	Ambiguous	0.22	Likely Benign	0.25	Likely Benign	-0.99	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.26	Benign	0.05	Affected			4	3	0.3	14.03
c.1559C>T	S520F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.541	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.833	Likely Pathogenic	-1.20	Ambiguous	0.4	0.39	Likely Benign	-0.41	Likely Benign	0.25	Likely Benign	-5.57	Deleterious	0.999	Probably Damaging	0.996	Probably Damaging	-1.36	Pathogenic	0.00	Affected	3.37	35	-2	-3	3.6	60.10
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.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.2168C>T	T723I (3D Viewer)	Likely Benign	GAP	Likely Benign		1	6-33441633-C-T	2	1.24e-6	-2.591	Likely Benign	0.120	Likely Benign	Likely Benign	0.045	Likely Benign	-0.39	Likely Benign	0.0	-0.20	Likely Benign	-0.30	Likely Benign	0.26	Likely Benign	-2.09	Neutral	0.088	Benign	0.030	Benign	3.39	Benign	0.03	Affected	3.50	8	0	-1	5.2	12.05	252.3	-31.6	0.0	0.0	-0.2	0.2						X		Uncertain	The hydroxyl group of Thr723, located on the outer surface of an α-helix (res. Leu714-Arg726), continuously forms hydrogen bonds with the backbone carbonyl of Asn719 in the WT simulations, potentially lowering the stability of the α-helix. In the variant simulations, the sec-butyl side chain of Ile723 cannot form any hydrogen bonds, which, in theory, could increase the helix stability. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.1712C>T	S571L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440764-C-T	1	6.23e-7	-11.651	Likely Pathogenic	0.660	Likely Pathogenic	Likely Benign	0.841	Likely Pathogenic	-1.53	Ambiguous	0.1	-1.05	Ambiguous	-1.29	Ambiguous	0.27	Likely Benign	-5.61	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	-1.25	Pathogenic	0.04	Affected	3.37	35	-2	-3	4.6	26.08
c.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.1925A>C	K642T (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-12.823	Likely Pathogenic	0.948	Likely Pathogenic	Ambiguous	0.484	Likely Benign	0.53	Ambiguous	0.1	0.30	Likely Benign	0.42	Likely Benign	0.28	Likely Benign	-5.88	Deleterious	0.872	Possibly Damaging	0.839	Possibly Damaging	2.86	Benign	0.00	Affected	3.37	31	0	-1	3.2	-27.07	213.5	-8.7	-0.3	0.4	0.3	0.2				X				Uncertain	The amino side chain of Lys642, located on the surface of an α helix (res. Ser641-Glu666), is not involved in any interactions in the WT simulations. In the variant simulations, the shorter side chain of Thr642 forms hydrogen bonds with Glu643 and Thr640 on the same α helix.Regardless, Lys642 is positioned directly at the GAP-Ras interface, and in the SynGAP-Ras WT simulations, its amino side chain forms salt bridges with the carboxylate groups of Ras residues Asp33 and Asp38. The shorter Thr642 is more likely to prefer hydrogen bonding with Glu643 and Thr640 on the same α helix, even in the Ras complex. Thus, the effect of the residue swap on the complex formation with the GTPase cannot be explored using solvent-only simulations.
c.2111G>C	S704T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.930	Likely Benign	0.265	Likely Benign	Likely Benign	0.071	Likely Benign	0.80	Ambiguous	0.0	0.15	Likely Benign	0.48	Likely Benign	0.29	Likely Benign	-1.72	Neutral	0.525	Possibly Damaging	0.107	Benign	3.45	Benign	0.07	Tolerated	3.47	10	1	1	0.1	14.03	201.7	-18.0	0.0	0.0	-0.2	0.7						X		Potentially Benign	Ser704 is located at the end and outer surface of an α-helix (res. Thr704-Gly712), which is connected via a tight turn or loop to another α-helix (res. Asp684-Gln702). The hydroxyl side chain of Ser704 occasionally forms a hydrogen bond with the amide group of Ala707. Similarly, in the variant simulations, the hydroxyl side chain of Thr704 forms hydrogen bonds with the amide groups of Ala707 and Leu708. Thus, the residue swap does not cause any apparent structural change.
c.745G>A	A249T (3D Viewer)	Likely Benign	PH	Uncertain		1				-3.564	Likely Benign	0.805	Likely Pathogenic	Ambiguous	0.487	Likely Benign	1.50	Ambiguous	0.6	1.39	Ambiguous	1.45	Ambiguous	0.30	Likely Benign	-0.96	Neutral	0.990	Probably Damaging	0.815	Possibly Damaging	5.65	Benign	0.40	Tolerated	3.39	15	1	0	-2.5	30.03	214.5	-43.3	0.0	0.0	0.5	0.2						X		Potentially Benign	The methyl group of Ala249, located on the surface of an α helix (res. Ala236-Val250) facing an anti-parallel β sheet strand (res. Ile205-Val209), packs against nearby hydrophobic residues such as Leu200, Leu246, and Val250. In the variant simulations, the hydroxyl group of Thr249, which is not suitable for hydrophobic packing, forms a stable hydrogen bond with the backbone carbonyl of Asn245 in the same helix. Although this interaction could theoretically weaken the structural integrity of the α helix, this destabilizing effect is not observed in the variant simulations.
c.1300G>A	V434I (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438205-G-A	1	6.19e-7	-6.999	Likely Benign	0.129	Likely Benign	Likely Benign	0.192	Likely Benign	-0.04	Likely Benign	0.0	0.22	Likely Benign	0.09	Likely Benign	0.31	Likely Benign	-0.82	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.53	Benign	0.18	Tolerated	3.37	29	4	3	0.3	14.03	246.7	-27.7	0.0	0.0	0.1	0.0						X		Potentially Benign	The iso-propyl side chain of Val434, located at the end of an α helix (res. Met414-Glu436), packs against hydrophobic residues in an interhelix space (e.g., Met430, Ala707, Leu711). In the variant simulations, the sec-butyl group of Ile434 is able to form the same hydrophobic interactions. Accordingly, the residue swap does not negatively affect the protein structure based on the simulations.
c.1976C>T	S659F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.925	Likely Pathogenic	0.662	Likely Pathogenic	Likely Benign	0.194	Likely Benign	-0.81	Ambiguous	0.1	-0.25	Likely Benign	-0.53	Ambiguous	0.32	Likely Benign	-4.59	Deleterious	0.806	Possibly Damaging	0.171	Benign	3.39	Benign	0.05	Affected	3.38	28	-3	-2	3.6	60.10	221.3	-61.2	0.0	0.0	0.6	0.4						X		Potentially Benign	In the WT simulations, the hydroxyl group of Ser659, located in a kink in the middle of the long α-helix (res. Ser641-Glu666), forms a hydrogen bond with the carboxylate group of Glu656. However, the phenol ring of the Phe659 side chain cannot form a similar hydrogen bond. Instead, it interacts with the hydrophobic isopropyl side chain of Val555 from the opposing α-helix (res. Ala533-Val560). This residue swap may therefore cause issues during protein folding.
c.878G>A	R293H	Likely Pathogenic	C2	Uncertain		1				-13.009	Likely Pathogenic	0.973	Likely Pathogenic	Likely Pathogenic	0.438	Likely Benign	4.45	Destabilizing	2.3	2.12	Destabilizing	3.29	Destabilizing	0.32	Likely Benign	-4.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.45	Pathogenic	0.04	Affected			2	0	1.3	-19.05
c.597C>A	N199K (3D Viewer)		PH	Uncertain		1				-8.198	Likely Pathogenic	0.686	Likely Pathogenic	Likely Benign	0.024	Likely Benign	-0.19	Likely Benign	0.1	0.03	Likely Benign	-0.08	Likely Benign	0.33	Likely Benign	-1.48	Neutral	0.276	Benign	0.083	Benign	4.27	Benign	0.13	Tolerated	3.47	9	1	0	-0.4	14.07	207.8	21.5	-0.1	1.5	0.1	0.0						X		Uncertain	Asn199, located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by a positively charged lysine. On the protein surface, both the carboxamide group of Asn199 and the amino group of Lys199 side chains can form hydrogen bonds with the backbone carbonyl groups of residues (e.g., Ala249) at the end of an α helix (res. Ala236-Lys251). However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.1405G>A	A469T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.540	Likely Pathogenic	0.723	Likely Pathogenic	Likely Benign	0.527	Likely Pathogenic	2.26	Destabilizing	0.1	1.90	Ambiguous	2.08	Destabilizing	0.34	Likely Benign	-1.46	Neutral	0.994	Probably Damaging	0.986	Probably Damaging	-1.21	Pathogenic	0.42	Tolerated			1	0	-2.5	30.03
c.886T>G	S296A (3D Viewer)	Likely Benign	C2	Uncertain		1				-6.847	Likely Benign	0.247	Likely Benign	Likely Benign	0.209	Likely Benign	0.50	Ambiguous	0.3	-0.26	Likely Benign	0.12	Likely Benign	0.35	Likely Benign	-1.79	Neutral	0.992	Probably Damaging	0.987	Probably Damaging	1.97	Pathogenic	0.65	Tolerated	3.40	16	1	1	2.6	-16.00	182.5	26.6	-0.2	0.1	-0.5	0.0		X						Potentially Pathogenic	The hydroxyl group of the Ser296 side chain, located in an anti-parallel β sheet strand (res. Met289-Pro298), stably hydrogen bonds with the carboxylate group of Asp330 in a neighboring β strand (res. Ala322-Asp332). The backbone carbonyl group of Ser296 also hydrogen bonds with the guanidinium group of Arg279 in another nearby β strand (res. Arg279-Cys285). In the variant simulations, the methyl group of the Ala296 side chain cannot hydrogen bond with Asp330, causing the carboxylate group positioning to fluctuate more than in the WT simulations.Although the residue swap does not seem to affect the anti-parallel β sheet assembly during the simulations, it is possible that the Ser296-Asp330 hydrogen bond plays a crucial role in maintaining the C2 domain fold. Notably, because Ser296 is located near the membrane interface, the potential effect of the residue swap on the SynGAP-membrane association cannot be addressed by solvent-only simulations.
c.1312G>A	A438T (3D Viewer)	Likely Benign	GAP	Conflicting		3	6-33438217-G-A	16	9.91e-6	-5.339	Likely Benign	0.085	Likely Benign	Likely Benign	0.021	Likely Benign	0.21	Likely Benign	0.0	-0.07	Likely Benign	0.07	Likely Benign	0.36	Likely Benign	-0.81	Neutral	0.300	Benign	0.011	Benign	4.18	Benign	0.14	Tolerated	3.38	26	1	0	-2.5	30.03	214.2	-42.7	-0.3	0.1	-0.4	0.1						X		Potentially Benign	The methyl group of Ala438, located in a four-residue loop connecting two α helices (res. Asn440-Thr458 and Pro413-Glu436), packs against hydrophobic residues from a nearby α helix or loop residues (e.g., Leu703, Val699). In the variant simulations, the methyl group of Thr438 is able to establish similar hydrophobic packing. Moreover, the hydroxyl group also H-bonds with nearby residues, such as the carbonyl group of the neighboring loop residue Pro437. Accordingly, the residue swap does not generate an apparent negative effect on the protein structure based on the simulations.
c.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.1404G>A	M468I (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438436-G-A	1	6.20e-7	-8.583	Likely Pathogenic	0.907	Likely Pathogenic	Ambiguous	0.508	Likely Pathogenic	2.53	Destabilizing	0.2	1.89	Ambiguous	2.21	Destabilizing	0.37	Likely Benign	-1.06	Neutral	0.748	Possibly Damaging	0.886	Possibly Damaging	-1.10	Pathogenic	0.07	Tolerated	3.37	31	1	2	2.6	-18.03
c.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.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.1082A>C	Q361P (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-13.280	Likely Pathogenic	0.956	Likely Pathogenic	Likely Pathogenic	0.482	Likely Benign	3.12	Destabilizing	0.0	3.45	Destabilizing	3.29	Destabilizing	0.38	Likely Benign	-3.03	Deleterious	0.996	Probably Damaging	0.979	Probably Damaging	1.63	Pathogenic	0.05	Affected	3.37	25	-1	0	1.9	-31.01
c.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.1610C>T	A537V (3D Viewer)	Likely Benign	GAP	Likely Benign		1	6-33438853-C-T	7	4.34e-6	-6.888	Likely Benign	0.120	Likely Benign	Likely Benign	0.382	Likely Benign	0.54	Ambiguous	0.0	-0.05	Likely Benign	0.25	Likely Benign	0.41	Likely Benign	-1.97	Neutral	0.977	Probably Damaging	0.469	Possibly Damaging	-1.26	Pathogenic	0.24	Tolerated	3.37	35	0	0	2.4	28.05	220.3	-45.1	0.0	0.0	-0.7	0.1						X		Potentially Benign	Ala537 is located on the outer surface of an α-helix (res. Ala533-Val560). The methyl group of Ala537 is on the surface and does not form any interactions. In the variant simulations, the iso-propyl side chain of Val537 is also on the surface, similar to Ala537 in the WT, causing no negative structural effects.
c.1456G>A	E486K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.545	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.435	Likely Benign	0.06	Likely Benign	0.1	0.37	Likely Benign	0.22	Likely Benign	0.41	Likely Benign	-3.58	Deleterious	1.000	Probably Damaging	0.988	Probably Damaging	3.40	Benign	0.12	Tolerated	3.37	35	0	1	-0.4	-0.94	206.8	52.1	-0.3	0.1	0.2	0.0	X			X				Uncertain	Glu486 is located in an α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. It is adjacent to the arginine finger (Arg485) and is expected to closely interact with Ras. The residue swap could affect complex formation with the GTPase and its activation. In the WT simulations, the carboxylate group of Glu486 forms salt bridges with Arg485 and Arg475 on the preceding α-helix (res. Ala461-Phe476). In the variant simulations, Lys486 does not form any specific interactions. Although the amino group of the Lys486 side chain cannot form these salt bridges, no negative effects on the protein structure are observed. Nevertheless, the potential role of Glu486 in SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations, and no definite conclusions can be drawn.
c.2015C>T	T672M (3D Viewer)		GAP	Conflicting		2	6-33441274-C-T	19	1.18e-5	-9.472	Likely Pathogenic	0.174	Likely Benign	Likely Benign	0.127	Likely Benign	0.31	Likely Benign	0.4	1.52	Ambiguous	0.92	Ambiguous	0.41	Likely Benign	-4.34	Deleterious	0.993	Probably Damaging	0.520	Possibly Damaging	3.39	Benign	0.00	Affected	3.40	25	-1	-1	2.6	30.09	231.9	-52.9	1.1	0.1	0.5	0.0		X				X		Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop itself, such as Lys566, Glu666, and Asn669. Met672 can only form a hydrogen bond with the amino group of the Lys566 side chain via its backbone carbonyl group. Nevertheless, the Lys566-Glu666 salt bridge forms intermittently. This is possible because Asn669 keeps the carboxylate group of Glu666 in the vicinity through hydrogen bonding, and the hydrophobic side chain of Met stays mostly rotated away from the salt bridge. Consequently, no drastic disruption of the hydrogen-bond network that keeps the loop close to the helices occurs in the variant simulations.
c.600G>C	L200F (3D Viewer)		PH	Uncertain		1	6-33435242-G-C	2	1.24e-6	-7.606	In-Between	0.592	Likely Pathogenic	Likely Benign	0.094	Likely Benign	1.00	Ambiguous	0.5	1.45	Ambiguous	1.23	Ambiguous	0.43	Likely Benign	-1.97	Neutral	0.997	Probably Damaging	0.916	Probably Damaging	4.02	Benign	0.17	Tolerated	3.46	9	2	0	-1.0	34.02	250.4	-15.1	0.6	0.2	0.5	0.0						X		Uncertain	Leu200, a hydrophobic residue located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by another hydrophobic residue, phenylalanine. Both the phenyl group of Phe200 and the branched iso-butyl hydrocarbon sidechain of Leu200 occupy an inward hydrophobic niche (e.g., Leu246, Val222, Phe231) during the simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.953C>T	P318L (3D Viewer)	Likely Pathogenic	C2	Uncertain		3	6-33437858-C-T	3	1.86e-6	-10.090	Likely Pathogenic	0.958	Likely Pathogenic	Likely Pathogenic	0.624	Likely Pathogenic	1.33	Ambiguous	0.1	0.26	Likely Benign	0.80	Ambiguous	0.43	Likely Benign	-8.96	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.82	Pathogenic	0.03	Affected	3.38	23	-3	-3	5.4	16.04	228.6	-68.9	-0.7	0.7	-0.4	0.1						X		Potentially Benign	The cyclic five-membered pyrrolidine ring of Pro318, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Asp330-Ala322, res. Thr305-Asn315), packs against the hydrophobic side chain of Ile205 at the end of the anti-parallel β sheet in the PH domain. In the variant simulations, the iso-butyl side chain of Leu318 is unable to do the same, potentially weakening the PH and C2 domain association. Importantly, the residue swap could also affect loop formation during folding, as proline can make tighter turns than leucine. Because the residue swap could affect the C2 domain stability, it could also negatively impact the SynGAP-membrane association.
c.1453C>T	R485C (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33438485-C-T	9	5.58e-6	-14.294	Likely Pathogenic	0.976	Likely Pathogenic	Likely Pathogenic	0.597	Likely Pathogenic	1.00	Ambiguous	0.1	0.26	Likely Benign	0.63	Ambiguous	0.44	Likely Benign	-7.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	1.90	Pathogenic	0.00	Affected	3.37	35	-4	-3	7.0	-53.05	225.5	99.6	-0.1	0.0	-0.3	0.2				X				Uncertain	The guanidinium group of Arg485 is located in a short helical structure (res. Glu480-Leu482) within an α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. The side chain of Arg485 acts as the “arginine finger” of SynGAP, playing a crucial role in Ras-GTPase activation. Consequently, the residue swap inhibits the conversion of GTP to GDP at the enzyme’s active site. Although no negative effects on the protein structure are observed during the simulations, no definite conclusions can be drawn due to the critical role of Arg485 in GTPase activation.
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.1055C>A	T352N (3D Viewer)	Likely Benign	C2	Likely Benign		1	6-33437960-C-A	2	1.24e-6	-4.817	Likely Benign	0.117	Likely Benign	Likely Benign	0.027	Likely Benign	0.20	Likely Benign	0.0	-0.04	Likely Benign	0.08	Likely Benign	0.45	Likely Benign	-0.92	Neutral	0.255	Benign	0.057	Benign	1.75	Pathogenic	0.19	Tolerated	3.37	25	0	0	-2.8	13.00	208.4	-14.5	-0.2	0.1	-0.1	0.0		X						Potentially Benign	Thr352 is located in a short α helical section within a loop connecting two β strands (res. Gly341-Pro349, res. Thr359-Pro364) originating from two different anti-parallel β sheets of the C2 domain. In the WT simulations, the side chain hydroxyl and backbone amide groups of Thr354 form hydrogen bonds with the backbone carbonyl group of Pro349 at the end of the preceding β strand. This arrangement likely stabilizes the α helical section and aids in folding, keeping the short secondary structure element intact in the variant simulations. However, the carboxamide group of the Asn352 side chain does not form hydrogen bonds with the backbone carbonyl group of Pro349. Instead, it packs against the cyclic ring and forms hydrogen bonds with the phenol group of the Tyr363 side chain in the other β strand.
c.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.1463C>T	T488M (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438495-C-T	2	1.24e-6	-12.459	Likely Pathogenic	0.973	Likely Pathogenic	Likely Pathogenic	0.746	Likely Pathogenic	0.66	Ambiguous	0.3	1.62	Ambiguous	1.14	Ambiguous	0.46	Likely Benign	-5.70	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	3.21	Benign	0.00	Affected	3.37	35	-1	-1	2.6	30.09
c.1973G>A	G658D (3D Viewer)		GAP	Uncertain		1	6-33441232-G-A	3	1.86e-6	-7.786	In-Between	0.442	Ambiguous	Likely Benign	0.144	Likely Benign	-0.40	Likely Benign	0.1	-0.59	Ambiguous	-0.50	Ambiguous	0.46	Likely Benign	-2.64	Deleterious	0.008	Benign	0.005	Benign	3.53	Benign	0.38	Tolerated	3.39	24	1	-1	-3.1	58.04	219.8	-84.3	0.0	0.0	0.2	0.1						X		Potentially Pathogenic	Gly658, located on the outer surface of an α helix (res. Ser641-Glu666), weakens the helix integrity at that spot, which is necessary for the kink in the middle of the long helix. In the variant simulations, the carboxylic acid side chain of Asp658 is on the surface of the α helix and is not involved in any interactions. However, aspartate is not as effective a breaker of the secondary structure element as glycine, which may lead to misfolding.
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.1169G>A	G390E (3D Viewer)		C2	Uncertain		1				-7.913	In-Between	0.646	Likely Pathogenic	Likely Benign	0.575	Likely Pathogenic	2.61	Destabilizing	0.9	4.28	Destabilizing	3.45	Destabilizing	0.47	Likely Benign	-0.87	Neutral	0.276	Benign	0.045	Benign	1.32	Pathogenic	0.05	Affected	4.32	8	0	-2	-3.1	72.06	241.5	-108.4	0.6	0.5	-0.1	0.1								Uncertain	Gly390 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364 and res. Ala399-Ile411). The Ω loop is assumed to directly interact with the membrane, and it is observed to move arbitrarily throughout the WT solvent simulations. This loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play significant roles in protein functions that require flexibility, and so they are rich in glycine residues, prolines, and to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Glu390 may not be as well tolerated in the Ω loop. Additionally, the carboxylate group of Glu390 occasionally forms H-bonds with other loop residues in the variant simulations. The interaction between the acidic carboxylate side chain and the acidic membrane lipids may further influence the SynGAP-membrane complex. However, since the effects on the Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1600T>C	S534P (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438843-T-C	3	1.86e-6	-5.056	Likely Benign	0.265	Likely Benign	Likely Benign	0.203	Likely Benign	-0.40	Likely Benign	0.2	0.35	Likely Benign	-0.03	Likely Benign	0.47	Likely Benign	-3.81	Deleterious	0.993	Probably Damaging	0.993	Probably Damaging	3.32	Benign	0.05	Affected	3.37	35	-1	1	-0.8	10.04
c.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.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.1436G>A	R479Q (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438468-G-A	7	4.34e-6	-7.109	In-Between	0.259	Likely Benign	Likely Benign	0.191	Likely Benign	0.54	Ambiguous	0.1	0.57	Ambiguous	0.56	Ambiguous	0.49	Likely Benign	-1.16	Neutral	1.000	Probably Damaging	0.991	Probably Damaging	3.42	Benign	0.31	Tolerated	3.39	32	1	1	1.0	-28.06
c.1195G>A	A399T (3D Viewer)	Likely Benign	C2	Benign		1				-5.236	Likely Benign	0.114	Likely Benign	Likely Benign	0.272	Likely Benign	1.24	Ambiguous	0.1	0.91	Ambiguous	1.08	Ambiguous	0.49	Likely Benign	-0.40	Neutral	0.131	Benign	0.039	Benign	5.41	Benign	0.69	Tolerated	3.38	26	1	0	-2.5	30.03	211.4	-41.4	0.0	0.0	0.6	0.4		X						Potentially Pathogenic	The methyl group of Ala399, located in an anti-parallel β sheet strand (res. Ala399-Ile411), is swapped for a hydroxyl-containing threonine. In the variant simulations, the hydroxyl group of Thr399 can form H-bonds with the backbone atoms of the residues in the membrane-facing loops (e.g., Gly382) in the C2 domain. Consequently, the ability of the Thr399 side chain to form H-bonds with the membrane-facing loops could adversely affect the dynamics and stability of the SynGAP-membrane association. However, since the effects on the dynamics of the membrane-facing loops can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.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.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.958G>C	V320L (3D Viewer)		C2	Uncertain		1	6-33437863-G-C	6	3.72e-6	-6.207	Likely Benign	0.362	Ambiguous	Likely Benign	0.096	Likely Benign	-0.26	Likely Benign	0.2	1.33	Ambiguous	0.54	Ambiguous	0.51	Ambiguous	-1.02	Neutral	0.900	Possibly Damaging	0.373	Benign	1.78	Pathogenic	0.92	Tolerated	3.38	23	2	1	-0.4	14.03	245.8	-10.2	0.3	0.9	0.1	0.3						X		Potentially Benign	The isopropyl side chain of Val310, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Thr305-Asn315, res. Ala322-Asp330), hydrophobically packs with the side chains of nearby residues (e.g., Leu286, Val350, Pro318). The hydrophobic Leu320 side chain mostly forms the same interactions; hence, the residue swap does not seem to negatively affect the protein structure based on the variant simulations.
c.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.1913A>G	K638R (3D Viewer)	Likely Benign	GAP	Uncertain		1				-2.700	Likely Benign	0.110	Likely Benign	Likely Benign	0.216	Likely Benign	0.09	Likely Benign	0.1	-0.04	Likely Benign	0.03	Likely Benign	0.53	Ambiguous	-2.55	Deleterious	0.649	Possibly Damaging	0.240	Benign	3.41	Benign	0.13	Tolerated	3.37	31	2	3	-0.6	28.01
c.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.1729G>A	A577T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33440781-G-A	6	3.72e-6	-5.311	Likely Benign	0.322	Likely Benign	Likely Benign	0.427	Likely Benign	0.86	Ambiguous	0.1	0.54	Ambiguous	0.70	Ambiguous	0.54	Ambiguous	-1.47	Neutral	0.999	Probably Damaging	0.987	Probably Damaging	-1.31	Pathogenic	0.47	Tolerated	3.37	34	1	0	-2.5	30.03	191.9	-43.4	0.0	0.0	0.7	0.1						X		Potentially Benign	Ala577 is located near the end and outer surface of an α-helix (res. Arg563-Glu578), where its methyl group does not form any particular interactions in the WT simulations. In the variant simulations, the hydroxyl group of the Thr577 side chain hydrogen bonds with the backbone atoms of Arg573 and Lys574 within the same helix, which has the potential to weaken the stability of the secondary structure element. Regardless, the residue swap seems to be well tolerated based on the variant simulations.
c.2111G>A	S704N (3D Viewer)	Likely Benign	GAP	Benign/Likely benign		3	6-33441370-G-A	27	1.67e-5	-5.917	Likely Benign	0.421	Ambiguous	Likely Benign	0.058	Likely Benign	0.48	Likely Benign	0.1	-0.12	Likely Benign	0.18	Likely Benign	0.54	Ambiguous	-0.49	Neutral	0.771	Possibly Damaging	0.275	Benign	3.39	Benign	0.08	Tolerated	3.47	10	1	1	-2.7	27.03	233.2	-29.1	-0.1	0.0	-0.1	0.1						X		Potentially Benign	Ser704 is located at the end and outer surface of an α-helix (res. Thr704-Gly712), which is connected via a tight turn or loop to another α-helix (res. Asp684-Gln702). The hydroxyl side chain of Ser704 occasionally forms a hydrogen bond with the amide group of Ala707. However, in the variant simulations, the carboxamide side chain of Asn704 achieves more lasting and numerous hydrogen-bonding interactions with the residues at the helix end, such as Glu706, Ala707, and Leu708. Consequently, the residue swap could strengthen the α-helix secondary structure integrity at the helix end, which could have either positive or negative effects on its function.
c.2050G>C	D684H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.194	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.613	Likely Pathogenic	3.36	Destabilizing	1.0	2.95	Destabilizing	3.16	Destabilizing	0.55	Ambiguous	-6.98	Deleterious	1.000	Probably Damaging	0.972	Probably Damaging	3.36	Benign	0.00	Affected	3.42	17	-1	1	0.3	22.05
c.1702G>T	V568L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.503	Likely Pathogenic	0.921	Likely Pathogenic	Ambiguous	0.651	Likely Pathogenic	-0.30	Likely Benign	0.3	0.57	Ambiguous	0.14	Likely Benign	0.56	Ambiguous	-2.69	Deleterious	0.511	Possibly Damaging	0.147	Benign	-1.23	Pathogenic	0.04	Affected	3.37	35	1	2	-0.4	14.03
c.1423C>T	R475W (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438455-C-T	1	6.20e-7	-13.235	Likely Pathogenic	0.962	Likely Pathogenic	Likely Pathogenic	0.725	Likely Pathogenic	1.44	Ambiguous	0.4	-0.92	Ambiguous	0.26	Likely Benign	0.56	Ambiguous	-7.56	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.45	Pathogenic	0.00	Affected	3.39	28	2	-3	3.6	30.03	266.9	39.6	0.0	0.0	0.0	0.1	X	X		X				Potentially Pathogenic	In the WT simulations, the guanidinium group of Arg475, located near the end of an α-helix (res. Ala461-Phe476), stacks with the phenyl ring of Phe476 and forms a salt bridge with Glu472. Additionally, Arg475 occasionally forms another salt bridge with the carboxylate group of Glu486 on the α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. Therefore, Arg475 potentially plays a key role in positioning the loop by interacting with Glu486, which is necessary for the positioning of the “arginine finger” (Arg485) and, ultimately, for RasGTPase activation.In the variant simulations, Trp475 moves and stacks with Arg479 on the proceeding α-α loop, disrupting the terminal end of the α-helix. Lastly, the potential effect of the residue swap on the SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.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.1835A>C	Q612P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.684	Likely Pathogenic	0.673	Likely Pathogenic	Likely Benign	0.671	Likely Pathogenic	-0.19	Likely Benign	0.3	3.06	Destabilizing	1.44	Ambiguous	0.56	Ambiguous	-5.84	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.31	Pathogenic	0.19	Tolerated			0	-1	1.9	-31.01
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.892C>T	P298S (3D Viewer)	Likely Benign	C2	Benign		1	6-33437797-C-T	5	3.10e-6	-6.342	Likely Benign	0.144	Likely Benign	Likely Benign	0.189	Likely Benign	1.38	Ambiguous	0.2	1.41	Ambiguous	1.40	Ambiguous	0.58	Ambiguous	-1.20	Neutral	0.991	Probably Damaging	0.898	Possibly Damaging	2.03	Pathogenic	0.85	Tolerated	3.39	20	-1	1	0.8	-10.04
c.962G>A	R321H (3D Viewer)		C2	Uncertain		1	6-33437867-G-A	8	4.96e-6	-8.751	Likely Pathogenic	0.136	Likely Benign	Likely Benign	0.323	Likely Benign	0.48	Likely Benign	0.1	-0.36	Likely Benign	0.06	Likely Benign	0.59	Ambiguous	-1.43	Neutral	1.000	Probably Damaging	0.998	Probably Damaging	1.92	Pathogenic	0.25	Tolerated	3.38	23	2	0	1.3	-19.05	218.5	86.9	1.1	0.0	0.3	0.0						X		Potentially Benign	The guanidinium group of Arg321, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Thr305-Asn315, res. Ala322-Asp330), faces outward without forming any stable interactions in the WT simulations. Similarly, in the variant simulations, the imidazole ring of His321 also points outward without making any stable intra-protein interactions. Thus, the residue swap does not seem to cause adverse effects on the protein structure based on the simulations. However, β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant.
c.1354G>T	V452F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.769	Likely Pathogenic	0.975	Likely Pathogenic	Likely Pathogenic	0.511	Likely Pathogenic	9.21	Destabilizing	0.1	0.37	Likely Benign	4.79	Destabilizing	0.61	Ambiguous	-4.94	Deleterious	0.999	Probably Damaging	0.993	Probably Damaging	3.29	Benign	0.00	Affected	3.37	34	-1	-1	-1.4	48.04	249.4	-35.7	0.0	0.0	0.4	0.1							X	Potentially Pathogenic	The iso-propyl side chain of Val452, located in the middle of an α helix (res. Val441-Ser457), packs against hydrophobic residues in the inter-helix space at the intersection of three α helices (e.g., Leu500, His453, Leu465). In the variant simulations, the larger side chain of Phe452 cannot pack against the opposing α helix (res. Leu489-Glu519) as efficiently as valine. Due to space restrictions, the phenol ring adjusts to make room by rotating slightly sideways in the inter-helix space. Besides this small and local shift, no large-scale effects on the protein structure are seen based on the simulations. However, the size difference between the swapped residues could affect the protein folding process.
c.694G>A	A232T (3D Viewer)		PH	Benign		1	6-33435545-G-A	1	6.20e-7	-7.655	In-Between	0.874	Likely Pathogenic	Ambiguous	0.469	Likely Benign	0.47	Likely Benign	0.1	-0.04	Likely Benign	0.22	Likely Benign	0.61	Ambiguous	-1.42	Neutral	0.608	Possibly Damaging	0.240	Benign	5.80	Benign	0.09	Tolerated	3.40	14	1	0	-2.5	30.03	210.8	-42.0	0.5	0.1	0.4	0.5						X		Uncertain	The hydroxyl group of Thr232, located at the end of an anti-parallel β sheet strand (res. Thr228-Ala232), forms hydrogen bonds with nearby residues Glu217, Cys233, and Cys219 in the variant simulations. These hydrogen-bonding interactions at the β sheet surface contribute to the stability of the secondary structure element and prevent it from unfolding. The new hydrogen bond interactions may be more favorable for structural stability than the steric interactions of the methyl side chain of Ala with the side chains of Gln216 and Cys219 in the WT. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.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.1640G>A	C547Y (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-15.871	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.874	Likely Pathogenic	8.53	Destabilizing	1.8	6.20	Destabilizing	7.37	Destabilizing	0.62	Ambiguous	-10.57	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.33	Pathogenic	0.06	Tolerated	3.37	35	0	-2	-3.8	60.04	280.1	-54.8	0.0	0.0	0.0	0.0		X	X				X	Potentially Pathogenic	Cys547 is located in an α-helix (res. Ala533-Val560). The thiol side chain of Cys547 is situated in a hydrophobic inter-helix space, where it packs hydrophobically with other residues such as Ile626, Leu551, and Phe652. Additionally, the thiol side chain of Cys weakly hydrogen bonds with the carbonyl group of Leu543 in the same α-helix. In the variant simulations, the bulkier phenol ring of Tyr547, with its polar hydroxyl group, is less suited for the hydrophobic space. Consequently, it moves outside and forms a hydrogen bond with the carbonyl group of Phe652 in the neighboring α-helix (res. Glu666-Asp644). This causes the two helices to slightly separate, negatively affecting the secondary structure integrity of the latter helix. These negative structural effects could be more pronounced during protein folding and are likely to be undermined in the MD simulations.
c.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.1635G>A	M545I (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.348	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.592	Likely Pathogenic	0.47	Likely Benign	0.1	0.14	Likely Benign	0.31	Likely Benign	0.63	Ambiguous	-3.61	Deleterious	0.935	Possibly Damaging	0.941	Probably Damaging	-1.27	Pathogenic	0.28	Tolerated	3.37	35	1	2	2.6	-18.03
c.859G>C	D287H (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-14.518	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.589	Likely Pathogenic	0.48	Likely Benign	0.3	0.32	Likely Benign	0.40	Likely Benign	0.63	Ambiguous	-6.43	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.51	Pathogenic	0.00	Affected	3.38	23	1	-1	0.3	22.05	235.6	3.8	0.1	1.2	0.1	0.1	X	X						Potentially Pathogenic	The carboxylate group of Asp287, located at the beginning of a β hairpin loop connecting two anti-parallel β sheet strands (res. Arg279-Leu286, res. Met289-Pro298), maintains a salt bridge with the guanidinium group of Arg324 in the β sheet during the WT simulations. In the variant simulations, the imidazole ring of the His287 side chain is unable to form a salt bridge with Arg324 or establish any other stable compensatory interactions, which could weaken the beta sandwich assembly of the C2 domain. This destabilization of the C2 domain could adversely affect the stability of the SynGAP-membrane association.
c.1888A>G	I630V (3D Viewer)		GAP	Benign/Likely benign		4	6-33440940-A-G	59	3.66e-5	-7.264	In-Between	0.145	Likely Benign	Likely Benign	0.143	Likely Benign	1.33	Ambiguous	0.0	0.94	Ambiguous	1.14	Ambiguous	0.64	Ambiguous	-0.38	Neutral	0.018	Benign	0.011	Benign	-1.37	Pathogenic	0.35	Tolerated	3.37	34	4	3	-0.3	-14.03	235.0	26.2	-0.1	0.0	-0.3	0.1						X		Potentially Benign	The sec-butyl side chain of Ile630, located in an α helix (res. Glu617-Asn635), packs with hydrophobic residues (e.g., Phe594, Leu633, Ile626, Ile602) in the hydrophobic inter-helix space between two α helices (res. Glu617-Asn635 and res. Glu582-Met603).In the variant simulations, the iso-propyl side chain of Val630, which shares a similar size and physicochemical properties with Ile630 in the WT, maintains similar interactions in the inter-helix space. Although no negative structural effects are observed during the simulations, the implications of the residue swap on the complex formation with the GTPase, due to its location, cannot be investigated using solvent-only simulations.
c.707C>T	A236V (3D Viewer)		PH	Benign/Likely benign		2	6-33435558-C-T	6	3.72e-6	-8.752	Likely Pathogenic	0.267	Likely Benign	Likely Benign	0.777	Likely Pathogenic	0.61	Ambiguous	0.2	1.08	Ambiguous	0.85	Ambiguous	0.64	Ambiguous	-3.55	Deleterious	0.981	Probably Damaging	0.446	Benign	5.79	Benign	0.03	Affected	3.40	14	0	0	2.4	28.05	213.8	-44.7	0.0	0.0	-0.2	0.2						X		Potentially Benign	The methyl side chain of Ala236, located on an α helix (residues Ala236-Val250) facing an anti-parallel β sheet strand (residues Ile205-Val209), interacts hydrophobically with nearby residues such as Arg239 and Phe218. In the variant simulations, the isopropyl branched hydrocarbon side chain of Val236 maintains similar hydrophobic interactions as alanine in the WT, with an overall arrangement remarkably similar to Ala236. The residue swap does not affect the protein structure based on the simulations.
c.2014A>G	T672A (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441273-A-G	3	1.86e-6	-6.524	Likely Benign	0.109	Likely Benign	Likely Benign	0.046	Likely Benign	0.51	Ambiguous	0.3	1.15	Ambiguous	0.83	Ambiguous	0.65	Ambiguous	-3.20	Deleterious	0.006	Benign	0.002	Benign	3.44	Benign	0.12	Tolerated	3.40	25	1	0	2.5	-30.03	188.5	42.5	-0.1	0.3	0.2	0.0		X						Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop itself, such as Lys566, Glu666, and Asn669. In the variant simulations, Ala672 can only form a hydrogen bond with Lys566 via its backbone carbonyl group. Consequently, it cannot maintain the Lys566-Glu666 salt bridge through hydrogen bonding, leading to a significant disruption of the intricate and stable hydrogen-bond network between the loop and the helices.
c.970C>T	R324W (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437875-C-T	2	1.24e-6	-12.906	Likely Pathogenic	0.694	Likely Pathogenic	Likely Benign	0.481	Likely Benign	1.49	Ambiguous	0.3	0.56	Ambiguous	1.03	Ambiguous	0.66	Ambiguous	-3.12	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.82	Pathogenic	0.16	Tolerated	3.39	22	2	-3	3.6	30.03	256.6	39.1	0.0	0.1	0.3	0.2		X						Potentially Pathogenic	The guanidinium group of Arg324, located at the end of an anti-parallel β sheet strand (res. Ala322-Asp330), faces outward and frequently forms a salt bridge with the carboxylate group of the Asp288 side chain, which is part of a β strand end (res. Met289-Pro298). In the variant simulations, the indole ring of the Trp324 side chain cannot maintain a similar interaction with the negatively charged carboxylate side chain of Asp288, potentially compromising the folding of the anti-parallel β sheet assembly. However, the residue swap does not appear to negatively impact the protein structure or its integrity based on the simulations.
c.1370G>A	S457N	Likely Pathogenic	GAP	Uncertain		1				-10.221	Likely Pathogenic	0.949	Likely Pathogenic	Ambiguous	0.241	Likely Benign	0.19	Likely Benign	0.0	-0.22	Likely Benign	-0.02	Likely Benign	0.67	Ambiguous	-2.76	Deleterious	0.940	Possibly Damaging	0.843	Possibly Damaging	3.28	Benign	0.06	Tolerated			1	1	-2.7	27.03
c.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.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.1651C>A	L551M (3D Viewer)		GAP	Uncertain		1	6-33438894-C-A	7	4.34e-6	-9.937	Likely Pathogenic	0.480	Ambiguous	Likely Benign	0.544	Likely Pathogenic	-0.07	Likely Benign	0.1	0.13	Likely Benign	0.03	Likely Benign	0.71	Ambiguous	-0.56	Neutral	1.000	Probably Damaging	1.000	Probably Damaging	-1.48	Pathogenic	0.06	Tolerated	3.37	35	4	2	-1.9	18.03	246.5	-18.6	0.0	0.0	0.3	0.0						X		Potentially Benign	L551 is located on an α-helix (res. Ala533-Val560). The iso-butyl side chain of Leu551 hydrophobically packs with nearby hydrophobic residues such as Cys547, Phe652, Leu633, and Ile630 in the inter-helix space. In the variant simulations, the thioether side chain of Met551 can maintain similar hydrophobic interactions as Leu551 in the WT, thus causing no negative effect on the protein structure during the simulations.
c.1768A>G	S590G (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2	6-33440820-A-G	14	8.67e-6	-14.277	Likely Pathogenic	0.574	Likely Pathogenic	Likely Benign	0.379	Likely Benign	0.67	Ambiguous	0.1	1.28	Ambiguous	0.98	Ambiguous	0.71	Ambiguous	-3.92	Deleterious	1.000	Probably Damaging	0.922	Probably Damaging	3.42	Benign	0.06	Tolerated	3.37	35	1	0	0.4	-30.03	186.7	49.4	0.0	0.0	0.1	0.0		X						Potentially Pathogenic	In the WT simulations, the hydroxyl group of Ser590, located on an α helix (res. Glu582-Met603), forms hydrogen bonds with the backbone carbonyl of Ala634 and/or the carboxamide group of the Asn635 side chain at the end of the opposing α helix (res. Thr619-Ala634).The residue swap could weaken the integrity of the α helix, as glycine is known as an “α helix breaker.” However, no discernible difference was observed between the WT and variant simulations in this regard. Importantly, Gly590 cannot form hydrogen bonds with the opposing helix in the same way that serine can, which could weaken the tertiary structure assembly between the two helices.
c.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.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.1222A>G	T408A		C2	Uncertain		1				-8.304	Likely Pathogenic	0.114	Likely Benign	Likely Benign	0.118	Likely Benign	0.37	Likely Benign	0.6	-0.06	Likely Benign	0.16	Likely Benign	0.72	Ambiguous	-3.07	Deleterious	0.540	Possibly Damaging	0.131	Benign	4.16	Benign	0.14	Tolerated			1	0	2.5	-30.03
c.1678G>A	V560M (3D Viewer)		GAP	Uncertain		2	6-33440730-G-A	15	9.50e-6	-9.598	Likely Pathogenic	0.517	Ambiguous	Likely Benign	0.520	Likely Pathogenic	-0.33	Likely Benign	0.1	0.88	Ambiguous	0.28	Likely Benign	0.72	Ambiguous	-2.42	Neutral	0.999	Probably Damaging	0.863	Possibly Damaging	-1.25	Pathogenic	0.14	Tolerated	3.37	35	2	1	-2.3	32.06	234.9	-52.6	0.0	0.0	-0.1	0.1						X		Potentially Benign	Val560 is located on the surface at the end of an α-helix (res. Ala533-Val560). The iso-propyl group of Val560 favorably packs against Asp508 of the opposing α-helix (res. Gln503-Glu519). However, in the variant simulations, the bulkier thioether side chain of Met560 does not form equally favorable inter-helix interactions. Regardless, no negative structural effects are observed during the simulations.
c.2015C>A	T672K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.192	Likely Pathogenic	0.698	Likely Pathogenic	Likely Benign	0.065	Likely Benign	0.20	Likely Benign	0.5	1.21	Ambiguous	0.71	Ambiguous	0.72	Ambiguous	-4.31	Deleterious	0.745	Possibly Damaging	0.051	Benign	3.40	Benign	0.07	Tolerated	3.40	25	0	-1	-3.2	27.07	195.1	7.0	0.4	0.7	0.4	0.1		X				X		Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop itself, such as Lys566, Glu666, and Asn669. In the variant simulations, Lys672 can only form a hydrogen bond with the amino group of the Lys566 side chain via its backbone carbonyl group. Consequently, it cannot maintain the Lys566-Glu666 salt bridge through hydrogen bonding. However, the amino group of Lys periodically forms a salt bridge with the carboxylate group of Glu666, which prevents a drastic disruption of the hydrogen-bond network that keeps the loop close to the helices.
c.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.667A>G	T223A (3D Viewer)		PH	Uncertain		1	6-33435518-A-G	3	1.86e-6	-7.076	In-Between	0.316	Likely Benign	Likely Benign	0.574	Likely Pathogenic	0.30	Likely Benign	0.1	0.77	Ambiguous	0.54	Ambiguous	0.74	Ambiguous	-3.36	Deleterious	0.231	Benign	0.058	Benign	5.74	Benign	0.09	Tolerated	3.41	13	1	0	2.5	-30.03	186.4	44.0	0.0	0.0	0.0	0.0	X	X						Uncertain	The introduced residue Ala223 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Unlike the hydroxyl group of the Thr223 side chain in the WT protein, the methyl side chain of Ala223 cannot form hydrogen bonds with nearby residues Thr228 and Lys207. Without these hydrogen-bonding interactions at the β sheet surface, the secondary structure element becomes unstable and partially unfolds in the variant simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.1428C>G	F476L (3D Viewer)		GAP	Uncertain		2	6-33438460-C-G	4	2.48e-6	-10.109	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.180	Likely Benign	1.00	Ambiguous	0.1	1.04	Ambiguous	1.02	Ambiguous	0.75	Ambiguous	-1.10	Neutral	0.997	Probably Damaging	0.978	Probably Damaging	3.53	Benign	0.60	Tolerated	3.40	22	2	0	1.0	-34.02	235.9	16.1	0.0	0.1	-0.2	0.0	X							Potentially Benign	In the WT simulations, the phenyl ring of Phe476, located at the end of an α-helix (res. Ala461-Phe476), packs with the hydrophobic side chains of Leu482 and Ile483. Additionally, Phe476 stacks with the Arg475 side chain on the preceding α-α loop connecting the two α-helices (res. Ala461-Phe476 and res. Leu489-Glu519) near the GAP-Ras interface.In the variant simulations, Leu476 can maintain hydrophobic packing with neighboring residues, although not as efficiently as the phenylalanine in the WT system. The absence of Phe476/Arg475 stacking weakens the integrity of the α-helix end in the variant simulations. Nonetheless, no large-scale adverse effects are observed in the simulations. Lastly, the potential effect of the residue swap on SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.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.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.1622C>G	A541G (3D Viewer)		GAP	Uncertain		1	6-33438865-C-G	2	1.24e-6	-7.233	In-Between	0.341	Ambiguous	Likely Benign	0.421	Likely Benign	0.67	Ambiguous	0.0	0.94	Ambiguous	0.81	Ambiguous	0.76	Ambiguous	-1.48	Neutral	0.999	Probably Damaging	0.995	Probably Damaging	-1.31	Pathogenic	0.57	Tolerated	3.37	35	1	0	-2.2	-14.03	170.1	23.6	0.0	0.0	0.0	0.0	X							Potentially Pathogenic	Ala541 is located on the outer surface of an α-helix (res. Ala533-Val560). The methyl group of Ala541 is on the surface and does not form any interactions. Glycine, known as an “α-helix breaker,” weakens the integrity of the helix. Indeed, in the variant simulations, the hydrogen bond formation between Gly541 and the backbone carbonyl of Ala537 is disrupted.
c.2047A>G	I683V (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441306-A-G	2	1.24e-6	-7.588	In-Between	0.138	Likely Benign	Likely Benign	0.112	Likely Benign	0.90	Ambiguous	0.0	0.60	Ambiguous	0.75	Ambiguous	0.76	Ambiguous	-0.78	Neutral	0.538	Possibly Damaging	0.080	Benign	3.35	Benign	0.14	Tolerated	3.42	17	4	3	-0.3	-14.03	215.6	29.1	0.0	0.0	-0.7	0.1						X		Potentially Benign	The sec-butyl side chain of Ile683, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is sterically packed against His453 and Glu688. In the variant simulations, the iso-propyl side chain of Val683 has similar size and physicochemical properties as Ile630 in the WT, and thus, it is able to maintain similar interactions in the inter-helix space. Consequently, no negative structural effects are observed during the simulations due to the residue swap.
c.895C>T	R299C (3D Viewer)	Likely Pathogenic	C2	Conflicting		2	6-33437800-C-T	3	1.86e-6	-6.326	Likely Benign	0.572	Likely Pathogenic	Likely Benign	0.344	Likely Benign	1.85	Ambiguous	0.4	0.61	Ambiguous	1.23	Ambiguous	0.76	Ambiguous	-3.54	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.65	Pathogenic	0.06	Tolerated	3.39	19	-4	-3	7.0	-53.05	210.7	91.3	0.1	0.0	0.0	0.2	X	X						Potentially Pathogenic	The guanidinium group of Arg299, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Met289-Pro298, res. Thr305-Asn315), forms hydrogen bonds that stabilize the tight turn. In the WT simulations, the Arg299 side chain hydrogen bonds with the loop backbone carbonyl groups (e.g., Ser302, Thr305, Leu274, Gly303), the hydroxyl group of Ser300, and even forms a salt bridge with the carboxylate group of Asp304.In the variant simulations, the thiol group of the Cys299 side chain is unable to form any of these well-coordinated or strong interactions, which could affect the initial formation of the secondary hairpin loop during folding. β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant. Moreover, the positively charged Arg299 side chain faces the polar head group region of the inner leaflet membrane and could directly anchor the C2 domain to the membrane. In short, the residue swap could negatively affect both protein folding and the stability of the SynGAP-membrane association.
c.913A>G	T305A (3D Viewer)	Likely Benign	C2	Conflicting		2	6-33437818-A-G	13	8.05e-6	-4.307	Likely Benign	0.078	Likely Benign	Likely Benign	0.144	Likely Benign	1.30	Ambiguous	0.6	1.55	Ambiguous	1.43	Ambiguous	0.77	Ambiguous	-2.10	Neutral	0.939	Possibly Damaging	0.645	Possibly Damaging	1.76	Pathogenic	0.12	Tolerated	3.40	20	1	0	2.5	-30.03	177.9	43.5	-0.2	0.1	0.4	0.0								Uncertain	The hydroxyl group of Thr305, located at the beginning of an anti-parallel β strand (res. Thr305-Asn315), hydrogen bonds with the carboxylate groups of Glu270 and Asp304 in the anti-parallel β strand and the adjacent β hairpin loop, respectively. In the variant simulations, the methyl group of the Ala305 side chain cannot hydrogen bond with either of the acidic residues, which could weaken the integrity of the tertiary structure and the β hairpin loop. Indeed, the guanidinium group of Arg299 does not acquire its central hairpin loop position due to the residue swap.β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant. Due to its location near the membrane surface, the residue swap could also affect the C2 loop dynamics and SynGAP-membrane association. However, this is beyond the scope of the solvent-only simulations to unravel.
c.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.1658A>C	K553T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.328	Likely Pathogenic	0.990	Likely Pathogenic	Likely Pathogenic	0.761	Likely Pathogenic	1.06	Ambiguous	0.2	0.48	Likely Benign	0.77	Ambiguous	0.79	Ambiguous	-5.77	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.34	Pathogenic	0.14	Tolerated	3.37	35	0	-1	3.2	-27.07	218.2	-10.7	0.0	0.0	-0.2	0.5		X						Potentially Pathogenic	Lys533 is located on an α-helix (res. Ala533-Val560). In the WT simulations, Lys533 packs against Phe513, and its amino side chain occasionally forms an ionic interaction with the carboxylate group of Glu512 from an opposing α-helix (res. Gln503-Tyr518). In the variant simulations, Thr533 is unable to reproduce these interactions, potentially weakening the integrity of the tertiary structure. Additionally, Thr533 forms a hydrogen bond with the backbone carbonyl group of Leu549 in the same helix, which could potentially weaken the secondary structure. Regardless, the residue swap does not cause significant structural effects based on the simulations.
c.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.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.1436G>C	R479P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.795	Likely Pathogenic	0.938	Likely Pathogenic	Ambiguous	0.277	Likely Benign	2.86	Destabilizing	0.2	3.88	Destabilizing	3.37	Destabilizing	0.81	Ambiguous	-3.52	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.41	Benign	0.18	Tolerated			0	-2	2.9	-59.07
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.1367A>C	Q456P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.250	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.469	Likely Benign	3.68	Destabilizing	0.2	8.43	Destabilizing	6.06	Destabilizing	0.82	Ambiguous	-5.66	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	3.34	Benign	0.07	Tolerated	3.37	34	-1	0	1.9	-31.01
c.1453C>A	R485S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.603	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.609	Likely Pathogenic	0.40	Likely Benign	0.1	1.07	Ambiguous	0.74	Ambiguous	0.82	Ambiguous	-5.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	1.93	Pathogenic	0.00	Affected			0	-1	3.7	-69.11
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.