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.3806T>A	V1269E	Likely Pathogenic	Coiled-coil	Uncertain		1				-11.418	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.403	Likely Benign										-5.05	Deleterious	0.999	Probably Damaging	0.995	Probably Damaging	2.09	Pathogenic	0.00	Affected	3.77	5	-2	-2	-7.7	29.98
c.3820C>T	R1274C			Uncertain		1	6-33447868-C-T			-6.467	Likely Benign	0.439	Ambiguous	Likely Benign	0.170	Likely Benign										-5.22	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	2.46	Pathogenic	0.00	Affected	3.77	5	-4	-3	7.0	-53.05
c.3821G>A	R1274H			Likely Benign		1	6-33447869-G-A	4	2.58e-6	-5.259	Likely Benign	0.256	Likely Benign	Likely Benign	0.149	Likely Benign										-3.20	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	2.49	Pathogenic	0.01	Affected	3.77	5	0	2	1.3	-19.05
c.3824G>A	R1275Q	Likely Benign		Uncertain		1	6-33447872-G-A	2	1.29e-6	-4.928	Likely Benign	0.121	Likely Benign	Likely Benign	0.103	Likely Benign										-1.72	Neutral	0.898	Possibly Damaging	0.147	Benign	2.59	Benign	0.03	Affected	3.77	5	1	1	1.0	-28.06
c.3824G>T	R1275L			Likely Benign		1	6-33447872-G-T	1	6.45e-7	-6.052	Likely Benign	0.446	Ambiguous	Likely Benign	0.117	Likely Benign										-4.04	Deleterious	0.800	Possibly Damaging	0.277	Benign	2.55	Benign	0.01	Affected	3.77	5	-3	-2	8.3	-43.03
c.3835G>A	A1279T	Likely Benign		Uncertain		2	6-33447883-G-A	2	1.29e-6	-4.871	Likely Benign	0.071	Likely Benign	Likely Benign	0.178	Likely Benign										-0.30	Neutral	0.001	Benign	0.000	Benign	2.71	Benign	0.09	Tolerated	3.77	5	1	0	-2.5	30.03
c.3846G>C	E1282D	Likely Benign		Uncertain		1	6-33447894-G-C	1	6.44e-7	-3.879	Likely Benign	0.074	Likely Benign	Likely Benign	0.104	Likely Benign										-1.26	Neutral	0.112	Benign	0.036	Benign	2.70	Benign	0.39	Tolerated	3.77	5	3	2	0.0	-14.03
c.3848C>T	P1283L	Likely Benign		Benign		1	6-33447896-C-T	32	2.06e-5	-3.740	Likely Benign	0.093	Likely Benign	Likely Benign	0.047	Likely Benign										-1.04	Neutral	0.005	Benign	0.003	Benign	2.76	Benign	0.06	Tolerated	3.77	5	-3	-3	5.4	16.04
c.3858A>T	E1286D	Likely Benign		Conflicting		4	6-33447906-A-T	143	9.22e-5	-4.010	Likely Benign	0.081	Likely Benign	Likely Benign	0.036	Likely Benign										1.02	Neutral	0.001	Benign	0.004	Benign	2.96	Benign	1.00	Tolerated	3.77	5	3	2	0.0	-14.03																10.1016/j.ajhg.2020.11.011
c.3859C>A	P1287T	Likely Benign		Uncertain		1	6-33447907-C-A			-3.940	Likely Benign	0.077	Likely Benign	Likely Benign	0.044	Likely Benign										-0.22	Neutral	0.126	Benign	0.041	Benign	2.78	Benign	0.04	Affected	3.77	5	-1	0	0.9	3.99
c.3860C>T	P1287L	Likely Benign		Conflicting		2	6-33447908-C-T			-2.800	Likely Benign	0.117	Likely Benign	Likely Benign	0.061	Likely Benign										-1.66	Neutral	0.021	Benign	0.017	Benign	2.76	Benign	0.02	Affected	3.77	5	-3	-3	5.4	16.04
c.3862A>G	K1288E			Uncertain		1	6-33447910-A-G	5	3.22e-6	-2.751	Likely Benign	0.407	Ambiguous	Likely Benign	0.185	Likely Benign										-3.27	Deleterious	0.979	Probably Damaging	0.973	Probably Damaging	2.13	Pathogenic	0.00	Affected	3.77	5	1	0	0.4	0.94
c.3902C>A	P1301H	Likely Benign		Conflicting		2	6-33451776-C-A	5	3.10e-6	-5.756	Likely Benign	0.104	Likely Benign	Likely Benign	0.232	Likely Benign										-1.13	Neutral	0.642	Possibly Damaging	0.378	Benign	2.79	Benign	0.04	Affected	3.77	5	0	-2	-1.6	40.02
c.3902C>G	P1301R	Likely Benign		Uncertain		1	6-33451776-C-G	15	9.30e-6	-4.753	Likely Benign	0.162	Likely Benign	Likely Benign	0.076	Likely Benign										-1.13	Neutral	0.077	Benign	0.059	Benign	2.81	Benign	0.10	Tolerated	3.77	5	0	-2	-2.9	59.07
c.3913A>G	T1305A	Likely Benign		Conflicting		4	6-33451787-A-G	30	1.86e-5	-2.692	Likely Benign	0.055	Likely Benign	Likely Benign	0.069	Likely Benign										1.74	Neutral	0.000	Benign	0.001	Benign	3.24	Benign	1.00	Tolerated	3.77	5	1	0	2.5	-30.03
c.391G>C	G131R			Uncertain		1				-6.564	Likely Benign	0.983	Likely Pathogenic	Likely Pathogenic	0.099	Likely Benign										-3.82	Deleterious	0.983	Probably Damaging	0.656	Possibly Damaging	3.92	Benign	0.00	Affected	3.61	5	-2	-3	-4.1	99.14
c.3920C>A	P1307Q	Likely Benign		Uncertain		1	6-33451794-C-A			-4.227	Likely Benign	0.114	Likely Benign	Likely Benign	0.192	Likely Benign										-0.88	Neutral	0.988	Probably Damaging	0.765	Possibly Damaging	2.82	Benign	0.03	Affected	3.77	5	0	-1	-1.9	31.01
c.3920C>T	P1307L	Likely Benign		Benign		1	6-33451794-C-T	11	6.82e-6	-4.044	Likely Benign	0.144	Likely Benign	Likely Benign	0.292	Likely Benign										-1.49	Neutral	0.779	Possibly Damaging	0.220	Benign	2.82	Benign	0.04	Affected	3.77	5	-3	-3	5.4	16.04
c.3922C>T	R1308C			Conflicting		2	6-33451796-C-T	4	2.48e-6	-4.994	Likely Benign	0.421	Ambiguous	Likely Benign	0.352	Likely Benign										-4.89	Deleterious	0.999	Probably Damaging	0.993	Probably Damaging	2.31	Pathogenic	0.00	Affected	3.77	5	-4	-3	7.0	-53.05
c.3923G>A	R1308H			Uncertain		1	6-33451797-G-A	3	1.86e-6	-3.586	Likely Benign	0.201	Likely Benign	Likely Benign	0.319	Likely Benign										-3.12	Deleterious	0.998	Probably Damaging	0.991	Probably Damaging	2.33	Pathogenic	0.00	Affected	3.77	5	2	0	1.3	-19.05
c.3929C>T	T1310M	Likely Benign		Benign		1	6-33451803-C-T	17	1.05e-5	-4.822	Likely Benign	0.117	Likely Benign	Likely Benign	0.069	Likely Benign										2.19	Neutral	0.021	Benign	0.005	Benign	2.98	Benign	0.93	Tolerated	3.77	5	-1	-1	2.6	30.09
c.3932T>C	L1311P	Likely Benign		Likely Benign		1	6-33451806-T-C	1	6.21e-7	-1.831	Likely Benign	0.079	Likely Benign	Likely Benign	0.123	Likely Benign										-0.52	Neutral	0.579	Possibly Damaging	0.335	Benign	2.72	Benign	0.18	Tolerated	3.77	5	-3	-3	-5.4	-16.04
c.3941C>T	P1314L	Likely Benign		Likely Benign		1	6-33451815-C-T	2	1.24e-6	-4.040	Likely Benign	0.118	Likely Benign	Likely Benign	0.049	Likely Benign										-0.20	Neutral	0.421	Benign	0.066	Benign	4.19	Benign	0.05	Affected	3.77	5	-3	-3	5.4	16.04
c.3943T>C	W1315R	Likely Benign		Uncertain		1				0.205	Likely Benign	0.660	Likely Pathogenic	Likely Benign	0.114	Likely Benign										1.31	Neutral	0.000	Benign	0.001	Benign	4.37	Benign	0.91	Tolerated	3.77	5	2	-3	-3.6	-30.03
c.3949G>A	G1317S	Likely Benign		Conflicting		3	6-33451823-G-A	1	6.26e-7	-3.522	Likely Benign	0.145	Likely Benign	Likely Benign	0.092	Likely Benign										-2.45	Neutral	0.127	Benign	0.045	Benign	4.08	Benign	0.00	Affected	3.77	5	1	0	-0.4	30.03
c.3956C>G	A1319G	Likely Benign		Uncertain		2	6-33451830-C-G			-3.927	Likely Benign	0.084	Likely Benign	Likely Benign	0.128	Likely Benign										-0.74	Neutral	0.819	Possibly Damaging	0.581	Possibly Damaging	4.07	Benign	0.06	Tolerated	3.77	5	1	0	-2.2	-14.03
c.3958C>T	P1320S	Likely Benign		Uncertain		1	6-33451832-C-T	2	1.28e-6	-4.928	Likely Benign	0.073	Likely Benign	Likely Benign	0.097	Likely Benign										-0.69	Neutral	0.980	Probably Damaging	0.968	Probably Damaging	4.25	Benign	0.00	Affected	3.77	5	1	-1	0.8	-10.04
c.3961C>T	P1321S	Likely Benign		Uncertain		2	6-33451835-C-T	10	6.46e-6	-4.897	Likely Benign	0.077	Likely Benign	Likely Benign	0.049	Likely Benign										0.68	Neutral	0.028	Benign	0.004	Benign	4.27	Benign	0.71	Tolerated	3.77	5	1	-1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.3962C>A	P1321Q	Likely Benign		Benign		1	6-33451836-C-A	1	6.58e-7	-5.594	Likely Benign	0.079	Likely Benign	Likely Benign	0.055	Likely Benign										-0.74	Neutral	0.659	Possibly Damaging	0.034	Benign	4.24	Benign	0.09	Tolerated	3.77	5	0	-1	-1.9	31.01
c.3964G>C	A1322P	Likely Benign		Benign		1	6-33451838-G-C			-1.153	Likely Benign	0.063	Likely Benign	Likely Benign	0.090	Likely Benign										0.03	Neutral	0.000	Benign	0.000	Benign	4.15	Benign	0.23	Tolerated	3.77	5	1	-1	-3.4	26.04
c.3977C>A	P1326Q	Likely Benign		Uncertain		1	6-33451851-C-A	1	6.40e-7	-5.422	Likely Benign	0.128	Likely Benign	Likely Benign	0.138	Likely Benign										-0.86	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	3.62	Benign	0.00	Affected	3.77	5	-1	0	-1.9	31.01
c.3977C>G	P1326R	Likely Benign		Uncertain		1				-5.097	Likely Benign	0.240	Likely Benign	Likely Benign	0.133	Likely Benign										-0.82	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	3.62	Benign	0.00	Affected	3.77	5	0	-2	-2.9	59.07
c.3977C>T	P1326L	Likely Benign		Uncertain		1				-5.541	Likely Benign	0.115	Likely Benign	Likely Benign	0.117	Likely Benign										-1.06	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	3.62	Benign	0.00	Affected	3.77	5	-3	-3	5.4	16.04
c.3979C>T	P1327S	Likely Benign		Uncertain		1	6-33451853-C-T			-4.744	Likely Benign	0.131	Likely Benign	Likely Benign	0.092	Likely Benign										0.28	Neutral	0.980	Probably Damaging	0.857	Possibly Damaging	4.25	Benign	0.71	Tolerated	3.77	5	1	-1	0.8	-10.04
c.3980C>T	P1327L	Likely Benign		Uncertain		1	6-33451854-C-T	2	1.28e-6	-5.264	Likely Benign	0.242	Likely Benign	Likely Benign	0.142	Likely Benign										-1.24	Neutral	0.994	Probably Damaging	0.908	Possibly Damaging	4.12	Benign	0.10	Tolerated	3.77	5	-3	-3	5.4	16.04
c.3983G>A	R1328Q	Likely Benign		Uncertain		3	6-33451857-G-A	35	1.49e-4	-2.921	Likely Benign	0.273	Likely Benign	Likely Benign	0.043	Likely Benign										-1.02	Neutral	0.799	Possibly Damaging	0.098	Benign	4.12	Benign	0.03	Affected	3.77	5	1	1	1.0	-28.06
c.3983G>C	R1328P	Likely Benign		Benign		1	6-33451857-G-C			-1.220	Likely Benign	0.466	Ambiguous	Likely Benign	0.060	Likely Benign										-2.01	Neutral	0.927	Possibly Damaging	0.452	Possibly Damaging	4.06	Benign	0.01	Affected	3.77	5	0	-2	2.9	-59.07
c.3995C>T	T1332M			Likely Benign		1	6-33451869-C-T	20	1.86e-5	-4.107	Likely Benign	0.948	Likely Pathogenic	Ambiguous	0.252	Likely Benign										-3.63	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	2.95	Benign	0.00	Affected	3.77	5	-1	-1	2.6	30.09
c.4000A>G	N1334D			Uncertain		1	6-33451874-A-G			-4.584	Likely Benign	0.674	Likely Pathogenic	Likely Benign	0.126	Likely Benign										-3.06	Deleterious	0.886	Possibly Damaging	0.522	Possibly Damaging	3.55	Benign	0.00	Affected	3.77	5	1	2	0.0	0.98
c.4003G>A	G1335S	Likely Pathogenic		Conflicting		2	6-33451877-G-A	3	2.37e-6	-4.495	Likely Benign	0.986	Likely Pathogenic	Likely Pathogenic	0.362	Likely Benign										-3.79	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	2.04	Pathogenic	0.00	Affected	3.77	5	1	0	-0.4	30.03
c.4006G>A	E1336K	Likely Benign		Benign		2	6-33451880-G-A	6	4.20e-6	-4.697	Likely Benign	0.977	Likely Pathogenic	Likely Pathogenic	0.272	Likely Benign										-2.44	Neutral	0.748	Possibly Damaging	0.079	Benign	3.23	Benign	0.00	Affected	3.77	5	0	1	-0.4	-0.94
c.4008G>C	E1336D	Likely Benign		Likely Benign		1				-3.344	Likely Benign	0.596	Likely Pathogenic	Likely Benign	0.062	Likely Benign										-1.92	Neutral	0.001	Benign	0.003	Benign	3.30	Benign	0.00	Affected	3.77	5	2	3	0.0	-14.03
c.4013G>A	R1338Q	Likely Benign		Conflicting		3	6-33451887-G-A	12	8.40e-6	-3.494	Likely Benign	0.317	Likely Benign	Likely Benign	0.076	Likely Benign										-1.87	Neutral	0.896	Possibly Damaging	0.194	Benign	3.81	Benign	0.02	Affected	3.77	5	1	1	1.0	-28.06
c.401G>A	S134N	Likely Benign		Uncertain		1				-5.534	Likely Benign	0.813	Likely Pathogenic	Ambiguous	0.075	Likely Benign										-1.62	Neutral	0.001	Benign	0.002	Benign	3.90	Benign	0.00	Affected	3.61	5	1	1	-2.7	27.03
c.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.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.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.2200C>T	P734S	Likely Benign		Uncertain		2	6-33441665-C-T	2	1.24e-6	-4.291	Likely Benign	0.077	Likely Benign	Likely Benign	0.030	Likely Benign										-2.44	Neutral	0.344	Benign	0.048	Benign	2.77	Benign	0.11	Tolerated	3.64	6	1	-1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.2291A>G	N764S	Likely Benign		Benign		1				-3.149	Likely Benign	0.159	Likely Benign	Likely Benign	0.058	Likely Benign										-0.84	Neutral	0.992	Probably Damaging	0.846	Possibly Damaging	2.65	Benign	0.61	Tolerated	3.64	6	1	1	2.7	-27.03
c.2294G>A	S765N	Likely Benign		Uncertain		1				-5.098	Likely Benign	0.378	Ambiguous	Likely Benign	0.094	Likely Benign										-0.94	Neutral	0.985	Probably Damaging	0.950	Probably Damaging	4.11	Benign	0.06	Tolerated	3.64	6	1	1	-2.7	27.03
c.2299A>G	I767V	Likely Benign		Uncertain		1				-2.791	Likely Benign	0.064	Likely Benign	Likely Benign	0.096	Likely Benign										0.10	Neutral	0.072	Benign	0.029	Benign	4.21	Benign	1.00	Tolerated	3.64	6	4	3	-0.3	-14.03
c.2300T>C	I767T	Likely Benign		Uncertain		1				-3.749	Likely Benign	0.252	Likely Benign	Likely Benign	0.138	Likely Benign										-0.78	Neutral	0.625	Possibly Damaging	0.249	Benign	4.12	Benign	0.46	Tolerated	3.64	6	0	-1	-5.2	-12.05
c.2302G>A	D768N	Likely Benign		Uncertain		1	6-33442460-G-A	2	2.57e-6	-6.892	Likely Benign	0.453	Ambiguous	Likely Benign	0.048	Likely Benign										-0.77	Neutral	0.106	Benign	0.009	Benign	4.07	Benign	0.96	Tolerated	3.64	6	1	2	0.0	-0.98
c.2302G>T	D768Y	Likely Pathogenic		Uncertain		1	6-33442460-G-T			-9.866	Likely Pathogenic	0.824	Likely Pathogenic	Ambiguous	0.234	Likely Benign										-2.86	Deleterious	0.989	Probably Damaging	0.806	Possibly Damaging	4.01	Benign	0.07	Tolerated	3.64	6	-4	-3	2.2	48.09
c.2324G>A	R775Q	Likely Benign		Conflicting		3	6-33442482-G-A	11	1.41e-5	-4.476	Likely Benign	0.229	Likely Benign	Likely Benign	0.085	Likely Benign										-0.63	Neutral	0.969	Probably Damaging	0.863	Possibly Damaging	4.17	Benign	0.16	Tolerated	3.64	6	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.2324G>C	R775P	Likely Benign		Benign		1				-5.072	Likely Benign	0.452	Ambiguous	Likely Benign	0.168	Likely Benign										-0.79	Neutral	0.971	Probably Damaging	0.944	Probably Damaging	4.13	Benign	0.07	Tolerated	3.64	6	-2	0	2.9	-59.07
c.2339C>G	S780C	Likely Benign		Uncertain		4	6-33442891-C-G	16	9.94e-6	-7.603	In-Between	0.278	Likely Benign	Likely Benign	0.078	Likely Benign										-1.41	Neutral	0.065	Benign	0.043	Benign	2.59	Benign	0.10	Tolerated	3.64	6	-1	0	3.3	16.06
c.2343G>A	M781I	Likely Benign		Benign		1				-2.484	Likely Benign	0.323	Likely Benign	Likely Benign	0.101	Likely Benign										0.05	Neutral	0.000	Benign	0.001	Benign	2.89	Benign	1.00	Tolerated	3.64	6	1	2	2.6	-18.03
c.2349G>A	M783I	Likely Benign		Benign		1	6-33442901-G-A	6	3.72e-6	-3.560	Likely Benign	0.418	Ambiguous	Likely Benign	0.042	Likely Benign										-0.54	Neutral	0.004	Benign	0.006	Benign	2.87	Benign	0.22	Tolerated	3.64	6	1	2	2.6	-18.03
c.2350G>A	A784T	Likely Benign		Benign		1				-3.579	Likely Benign	0.089	Likely Benign	Likely Benign	0.046	Likely Benign										1.23	Neutral	0.001	Benign	0.006	Benign	2.92	Benign	1.00	Tolerated	3.64	6	1	0	-2.5	30.03
c.2353C>T	R785C	Likely Pathogenic	SH3-binding motif	Uncertain		1	6-33442905-C-T	29	1.80e-5	-5.887	Likely Benign	0.662	Likely Pathogenic	Likely Benign	0.126	Likely Benign										-5.06	Deleterious	0.144	Benign	0.046	Benign	2.22	Pathogenic	0.00	Affected	3.64	6	-4	-3	7.0	-53.05
c.2354G>A	R785H		SH3-binding motif	Uncertain		2	6-33442906-G-A	4	2.50e-6	-4.782	Likely Benign	0.388	Ambiguous	Likely Benign	0.129	Likely Benign										-2.61	Deleterious	0.999	Probably Damaging	0.947	Probably Damaging	2.25	Pathogenic	0.01	Affected	3.64	6	2	0	1.3	-19.05
c.2359C>A	P787T	Likely Pathogenic	SH3-binding motif	Likely Benign		1	6-33442911-C-A	17	1.05e-5	-4.813	Likely Benign	0.603	Likely Pathogenic	Likely Benign	0.258	Likely Benign										-4.40	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.46	Pathogenic	0.01	Affected	3.64	6	0	-1	0.9	3.99
c.2359C>T	P787S		SH3-binding motif	Uncertain		1	6-33442911-C-T	3	1.86e-6	-4.203	Likely Benign	0.564	Ambiguous	Likely Benign	0.221	Likely Benign										-3.81	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.48	Pathogenic	0.02	Affected	3.64	6	-1	1	0.8	-10.04
c.2362T>A	S788T	Likely Benign	SH3-binding motif	Uncertain		2	6-33442914-T-A	4	2.49e-6	-4.288	Likely Benign	0.288	Likely Benign	Likely Benign	0.092	Likely Benign										-2.25	Neutral	0.979	Probably Damaging	0.982	Probably Damaging	1.55	Pathogenic	0.02	Affected	3.64	6	1	1	0.1	14.03
c.2369C>A	T790N		SH3-binding motif	Conflicting		3	6-33442921-C-A	69	4.28e-5	-5.243	Likely Benign	0.276	Likely Benign	Likely Benign	0.103	Likely Benign										-2.54	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.27	Pathogenic	0.02	Affected	3.64	6	0	0	-2.8	13.00
c.2369C>G	T790S	Likely Benign	SH3-binding motif	Uncertain		1				-3.914	Likely Benign	0.123	Likely Benign	Likely Benign	0.134	Likely Benign										-1.83	Neutral	0.997	Probably Damaging	0.989	Probably Damaging	2.39	Pathogenic	0.33	Tolerated	3.64	6	1	1	-0.1	-14.03
c.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.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.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.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.2195G>A	R732K	Likely Benign		Conflicting		2	6-33441660-G-A	4	2.48e-6	-5.278	Likely Benign	0.240	Likely Benign	Likely Benign	0.045	Likely Benign										-0.82	Neutral	0.973	Probably Damaging	0.943	Probably Damaging	2.69	Benign	0.21	Tolerated	3.59	7	3	2	0.6	-28.01
c.2195G>C	R732T			Uncertain		1				-8.545	Likely Pathogenic	0.434	Ambiguous	Likely Benign	0.075	Likely Benign										-1.96	Neutral	0.999	Probably Damaging	0.892	Possibly Damaging	2.59	Benign	0.12	Tolerated	3.59	7	-1	-1	3.8	-55.08
c.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.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.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.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.2131C>G	L711V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33441596-C-G	1	6.20e-7	-10.045	Likely Pathogenic	0.709	Likely Pathogenic	Likely Benign	0.170	Likely Benign	3.48	Destabilizing	0.1	2.22	Destabilizing	2.85	Destabilizing	1.40	Destabilizing	-2.59	Deleterious	0.992	Probably Damaging	0.970	Probably Damaging	3.34	Benign	0.00	Affected	3.50	9	1	2	0.4	-14.03
c.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.2143C>T	P715S (3D Viewer)		GAP	Likely Pathogenic		1	6-33441608-C-T	1	6.20e-7	-7.635	In-Between	0.787	Likely Pathogenic	Ambiguous	0.277	Likely Benign	3.54	Destabilizing	0.0	0.81	Ambiguous	2.18	Destabilizing	0.94	Ambiguous	-7.17	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.43	Benign	0.01	Affected	3.50	9	1	-1	0.8	-10.04	231.8	-14.0	-0.1	0.0	-0.8	0.1							X	Uncertain	Pro715, along with Gly712 and Pro713, are located in a hinge region of an α-helix making a ~90-degree turn (res. Lys705-Leu725). In the WT simulations, the pyrrolidine side chain of Pro715, lacking the backbone amide groups altogether, forces the tight helix turn to take place while also hydrophobically packing with nearby residues (e.g., Leu700, Leu708, Leu714, and Leu718). Leu715, with a normal amide backbone, could potentially affect protein folding and turn formation, although this was not observed in the variant simulations. Additionally, the hydroxyl group of the Ser715 side chain can form hydrogen bonds with the backbone carbonyl group of Gly712 and disrupt the hydrophobic packing arrangement of the leucine residues from the neighboring α-helices, impacting the GAP domain tertiary assembly.
c.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.2162T>G	I721S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.032	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.466	Likely Benign	3.91	Destabilizing	0.1	3.96	Destabilizing	3.94	Destabilizing	2.28	Destabilizing	-5.26	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.21	Pathogenic	0.00	Affected	3.50	9	-1	-2	-5.3	-26.08	203.3	49.3	-0.1	0.0	-1.1	0.0							X	Uncertain	The sec-butyl side chain of Ile721, located on an α-helix (res. Leu714-Arg726), engages in hydrophobic packing with other residues in the hydrophobic inter-helix space, such as Phe420, Tyr417, His693, and Leu717. In the variant simulations, the hydroxyl side chain of Ser721 forms hydrogen bonds with nearby residues, such as Leu717 and His693. Although no major structural changes are observed during the variant simulations, the hydrophilic residue Ser721 could disrupt the hydrophobic packing during folding. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
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.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.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.2095G>A	V699M (3D Viewer)		GAP	Uncertain		2	6-33441354-G-A	8	4.96e-6	-8.869	Likely Pathogenic	0.484	Ambiguous	Likely Benign	0.276	Likely Benign	-0.58	Ambiguous	0.1	0.29	Likely Benign	-0.15	Likely Benign	0.96	Ambiguous	-2.18	Neutral	0.994	Probably Damaging	0.806	Possibly Damaging	3.37	Benign	0.03	Affected	3.47	10	2	1	-2.3	32.06	257.8	-47.2	0.0	0.0	0.9	0.1						X		Potentially Benign	The isopropyl side chain of Val699, located on an α-helix (res. Leu685-Gln702), packs against hydrophobic residues (e.g., Leu703, Leu696, Leu435, Leu439) in the inter-helix space. In the variant simulations, the thioether side chain of Met699 has similar physicochemical properties to Val699 in the WT, and thus, it is able to maintain similar interactions. Consequently, the mutation causes no apparent changes in the structure.
c.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.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.2113A>C	K705Q (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441372-A-C	1	6.20e-7	-5.787	Likely Benign	0.436	Ambiguous	Likely Benign	0.142	Likely Benign	-0.10	Likely Benign	0.0	0.33	Likely Benign	0.12	Likely Benign	-0.02	Likely Benign	-0.24	Neutral	0.997	Probably Damaging	0.969	Probably Damaging	3.42	Benign	0.78	Tolerated	3.47	10	1	1	0.4	-0.04
c.2115G>C	K705N (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-9.767	Likely Pathogenic	0.925	Likely Pathogenic	Ambiguous	0.183	Likely Benign	0.74	Ambiguous	0.0	0.37	Likely Benign	0.56	Ambiguous	0.44	Likely Benign	-3.12	Deleterious	0.996	Probably Damaging	0.876	Possibly Damaging	3.37	Benign	0.02	Affected	3.47	10	1	0	0.4	-14.07	221.4	-20.2	0.0	0.0	0.0	0.1						X		Uncertain	The amino side chain of Lys705, located at the end and outer surface of an α-helix (res. Thr704-Gly712), does not form any interactions in the WT simulations. In the variant simulations, the carboxamide side chain of Asn705 briefly forms a salt bridge with Glu706. However, there is no apparent difference between the systems. Due to the model ending abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.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.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.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.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.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.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.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.680G>A	G227E (3D Viewer)	Likely Pathogenic	PH	Conflicting		2	6-33435531-G-A	3	1.86e-6	-9.186	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.792	Likely Pathogenic	2.56	Destabilizing	0.4	5.36	Destabilizing	3.96	Destabilizing	0.94	Ambiguous	-6.49	Deleterious	0.906	Possibly Damaging	0.360	Benign	5.72	Benign	0.01	Affected	3.43	12	0	-2	-3.1	72.06	237.7	-112.1	0.1	0.3	0.0	0.3		X				X		Uncertain	The introduced residue Glu227 is located in a β hairpin loop connecting two anti-parallel β sheet strands (res. Cys219-Thr224 and Thr228-Ala232). In the variant simulations, the carboxylate group of Glu227 frequently forms a salt bridge with the amino group of the neighboring residue Lys229. Despite this interaction, the integrity of the secondary structure element is not compromised. However, the β hairpins are potential nucleation sites during the initial stages of protein folding. Additionally, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.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.2086C>G	L696V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.909	Likely Pathogenic	0.745	Likely Pathogenic	Likely Benign	0.351	Likely Benign	2.35	Destabilizing	0.1	1.85	Ambiguous	2.10	Destabilizing	1.46	Destabilizing	-2.79	Deleterious	0.992	Probably Damaging	0.970	Probably Damaging	3.16	Benign	0.00	Affected	3.46	13	1	2	0.4	-14.03
c.2087T>C	L696P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-16.926	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.678	Likely Pathogenic	6.66	Destabilizing	0.2	10.84	Destabilizing	8.75	Destabilizing	2.13	Destabilizing	-6.58	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.00	Benign	0.00	Affected	3.46	13	-3	-3	-5.4	-16.04	180.6	65.9	0.1	0.0	-0.6	0.1	X							Potentially Pathogenic	The isobutyl side chain of Leu696, located in the middle of an α-helix (res. Leu685-Gln702), engages in hydrophobic packing with nearby residues (e.g., Leu441, Leu431, Leu692, Leu714) in the inter-helix space. Prolines lack a free amide group necessary for hydrogen bonding with the carbonyl group of Leu692 in the same manner as Leu696 in the WT. Consequently, the residue swap with proline disrupts the continuity of the secondary structure element in the variant simulations. Additionally, the side chain of Pro696 is not as optimal as Leu696 for hydrophobic packing in the inter-helix space.
c.2089T>C	W697R (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441348-T-C	1	6.20e-7	-10.020	Likely Pathogenic	0.941	Likely Pathogenic	Ambiguous	0.401	Likely Benign	1.14	Ambiguous	0.1	1.18	Ambiguous	1.16	Ambiguous	1.25	Destabilizing	-9.50	Deleterious	1.000	Probably Damaging	0.994	Probably Damaging	3.45	Benign	0.02	Affected	3.46	13	2	-3	-3.6	-30.03	254.4	-41.2	0.0	0.0	-0.7	0.0						X		Potentially Benign	The indole ring of Trp697, located on the outer surface of an α-helix (res. Leu685-Val699), is not involved in any long-lasting interactions in the WT simulations. In the variant simulations, the positively charged guanidinium side chain of Arg697 occasionally forms hydrogen bonds with nearby residues, such as Ser722 and Asn719. However, similar to Trp697 in the WT, Arg697 does not form any long-lasting interactions and thus does not induce any negative structural effects in the simulations.
c.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.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.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.670A>G	T224A (3D Viewer)		PH	Uncertain		3	6-33435521-A-G	2	1.24e-6	-7.379	In-Between	0.651	Likely Pathogenic	Likely Benign	0.464	Likely Benign	0.33	Likely Benign	0.1	1.05	Ambiguous	0.69	Ambiguous	0.91	Ambiguous	-2.96	Deleterious	0.243	Benign	0.079	Benign	5.57	Benign	0.57	Tolerated	3.41	13	1	0	2.5	-30.03	169.0	41.4	-0.5	1.1	-0.4	0.0	X	X						Uncertain	The introduced residue Ala224 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Unlike the hydroxyl group of the Thr224 side chain in the WT model, the methyl side chain of Ala224 cannot form hydrogen bonds with nearby residues Ser204, Ser226, and Gly227. Without these hydrogen-bonding interactions at the β sheet surface, the secondary structure element becomes unstable and unfolds during the variant simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.2071A>C	T691P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-13.801	Likely Pathogenic	0.905	Likely Pathogenic	Ambiguous	0.214	Likely Benign	5.04	Destabilizing	0.4	6.09	Destabilizing	5.57	Destabilizing	1.27	Destabilizing	-3.43	Deleterious	1.000	Probably Damaging	0.952	Probably Damaging	3.43	Benign	0.06	Tolerated	3.43	14	0	-1	-0.9	-3.99	188.9	33.0	0.1	0.0	-0.6	0.0	X	X						Potentially Pathogenic	The hydroxyl side chain of Thr691, located in an α-helix (res. Leu696-Leu685), can form hydrogen bonds with the backbone carbonyl and the side chain guanidinium group of Arg687. This interaction facilitates the simultaneous formation of salt bridges between Arg687 and Glu688 on the same α-helix. Additionally, Thr691 occasionally interacts with the thioether side chain of Met409 in an anti-parallel β-sheet of the C2 domain (res. Ile411-Ala399), although this interaction is not consistently maintained throughout the WT simulations. In the variant simulations, the pyrrolidine side chain of Pro691 lacks hydrogen bond donors, making a similar setup impossible. Moreover, proline lacks a free amide group necessary for hydrogen bonding with the carbonyl group of Arg687, introducing a slight bend in the α-helix and compromising its integrity.
c.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.700C>T	R234W (3D Viewer)	Likely Pathogenic	PH	Uncertain		1	6-33435551-C-T	3	1.86e-6	-12.625	Likely Pathogenic	0.947	Likely Pathogenic	Ambiguous	0.805	Likely Pathogenic	0.96	Ambiguous	0.3	0.69	Ambiguous	0.83	Ambiguous	0.13	Likely Benign	-5.52	Deleterious	0.997	Probably Damaging	0.803	Possibly Damaging	5.76	Benign	0.01	Affected	3.40	14	2	-3	3.6	30.03	262.8	39.6	-0.1	0.0	-0.2	0.2		X						Potentially Pathogenic	The guanidinium group of Arg234, located in a β-α loop between an anti-parallel β sheet strand (residues Gly227-Phe231) and an α helix (res. Ala236-Val250), forms a salt bridge with the carboxylate group of Glu238 in the α helix. Occasionally, it also bonds with the GAP domain residues Ser678 and Glu680. Thus, the positively charged Arg234 could contribute to the tertiary structure assembly between the PH and GAP domains. In contrast, the indole side chain of Trp234 in the variant is located on the protein surface in the variant simulations and is unable to form any interactions.
c.703T>C	S235P (3D Viewer)	Likely Pathogenic	PH	Likely Pathogenic		1				-14.857	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.870	Likely Pathogenic	4.02	Destabilizing	0.1	6.91	Destabilizing	5.47	Destabilizing	1.23	Destabilizing	-4.24	Deleterious	0.917	Possibly Damaging	0.446	Benign	5.47	Benign	0.01	Affected	3.40	14	1	-1	-0.8	10.04	201.5	17.0	0.1	0.0	-0.6	0.0		X						Potentially Pathogenic	In the WT, the hydroxyl group of Ser235, located in a β-α loop between an anti-parallel β sheet strand (res. Gly227-Phe231) and an α helix (residues Ala236-Val250), forms hydrogen bonds with the GAP domain loop residue Glu680 and with the backbone amide groups of Ala237 and Glu238 from the α helix. In the variant simulations, the pyrrolidine ring of Pro235 cannot stabilize the α helix end or maintain tertiary bonding interactions between the PH and GAP domains via hydrogen bonding as effectively as serine.
c.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.742C>T	R248W (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-11.647	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.699	Likely Pathogenic	1.17	Ambiguous	0.3	-0.20	Likely Benign	0.49	Likely Benign	0.89	Ambiguous	-6.98	Deleterious	1.000	Probably Damaging	0.948	Probably Damaging	5.62	Benign	0.00	Affected	3.41	14	2	-3	3.6	30.03	266.4	42.3	0.0	0.0	0.3	0.1		X						Potentially Pathogenic	The guanidinium group of Arg248, located on an α helix (res. Ala236-Val250), forms two very stable salt bridges with Asp255 (from a short α helical section, res. Lys254-Asn256) and Glu244 (from a nearby loop) in the WT simulations. In the variant simulations, the indole group of Trp248 cannot form any salt bridges, which could negatively affect the tertiary structure assembly of the PH domain. Instead, in the variant simulations, the indole ring of Trp248 stacks against Pro252, which makes a turn after the α helix.
c.743G>C	R248P (3D Viewer)	Likely Pathogenic	PH	Likely Pathogenic		1				-10.751	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.848	Likely Pathogenic	3.09	Destabilizing	0.6	8.87	Destabilizing	5.98	Destabilizing	1.21	Destabilizing	-5.97	Deleterious	0.998	Probably Damaging	0.878	Possibly Damaging	5.64	Benign	0.00	Affected	3.41	14	0	-2	2.9	-59.07	223.8	126.6	0.0	0.0	-0.2	0.1	X	X						Potentially Pathogenic	The guanidinium group of Arg248, located on an α helix (residues Ala236-Val250), forms two very stable salt bridges with Asp255 (from a short α helical section, res. Lys254-Asn256) and Glu244 (from a nearby loop) in the WT simulations. In the variant simulations, the pyrrolidine side chain of Pro248 cannot form any salt bridges, which could negatively affect the tertiary structure assembly of the PH domain. Additionally, Pro248 lacks a free amide group needed for hydrogen bonding with the backbone carbonyl group of Asn245, disrupting the continuity of the α helix.
c.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.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.772C>T	R258C (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437677-C-T	1	6.20e-7	-10.285	Likely Pathogenic	0.790	Likely Pathogenic	Ambiguous	0.771	Likely Pathogenic	1.17	Ambiguous	0.4	1.76	Ambiguous	1.47	Ambiguous	0.87	Ambiguous	-6.79	Deleterious	1.000	Probably Damaging	0.993	Probably Damaging	5.77	Benign	0.00	Affected	3.39	15	-3	-4	7.0	-53.05
c.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.762G>C	K254N (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-13.306	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.757	Likely Pathogenic	0.73	Ambiguous	0.2	1.87	Ambiguous	1.30	Ambiguous	1.19	Destabilizing	-4.23	Deleterious	0.384	Benign	0.070	Benign	5.93	Benign	0.01	Affected	3.39	15	1	0	0.4	-14.07	215.3	-21.0	-1.0	1.7	0.2	0.3		X						Potentially Pathogenic	The amino group of Lys254, located in an α-β loop connecting the PH and C2 domains (res. Lys251-Arg258), forms salt bridges with the carboxylate groups of Glu244 and Asp684. Since the neutral carboxamide group of the Asn254 side chain cannot form salt bridges with acidic residues, the residue swap potentially weakens the tertiary structure assembly and/or influences the loop positioning. Regardless, in both the variant and WT simulations, all hydrogen bonds formed by the residue’s side chain were broken, and the residue rotated outwards. The partially α helical conformation of the loop, which extends to a nearby α helix (res. Met414-Asn426), is dynamic, making it unclear if the mutation affects it.
c.773G>A	R258H (3D Viewer)		C2	Benign/Likely benign		3	6-33437678-G-A	10	6.20e-6	-10.533	Likely Pathogenic	0.525	Ambiguous	Likely Benign	0.830	Likely Pathogenic	1.60	Ambiguous	0.6	1.00	Ambiguous	1.30	Ambiguous	1.47	Destabilizing	-4.06	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	5.77	Benign	0.01	Affected	3.39	15	2	0	1.3	-19.05	212.5	81.8	0.1	0.0	-0.5	0.2		X						Potentially Pathogenic	The guanidinium group of Arg258, located at the end of an α-β loop connecting the PH domain to the C2 domain (res. Lys251-Arg258), forms hydrogen bonds with the carboxamide groups of Asn727 and Asn729 side chains, as well as with the backbone carbonyl groups of Ala724, Leu725, and Asn727 in the WT simulations. Although the imidazole group of His258 can act as a hydrogen bond donor/acceptor, the swapped residue is unable to maintain an equally well-coordinated hydrogen bond network for linking the C2 and GAP domains in the variant simulations.
c.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.986G>A	R329H (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437891-G-A	2	1.24e-6	-10.154	Likely Pathogenic	0.769	Likely Pathogenic	Likely Benign	0.155	Likely Benign	2.53	Destabilizing	0.7	0.71	Ambiguous	1.62	Ambiguous	0.82	Ambiguous	-3.17	Deleterious	0.995	Probably Damaging	0.778	Possibly Damaging	4.04	Benign	0.05	Affected	3.41	15	2	0	1.3	-19.05	220.4	81.4	0.1	0.1	0.2	0.3								Uncertain	The guanidinium group of Arg329, located at the end of an anti-parallel β sheet strand (res. Ala322-Asp330), faces the negatively charged lipid bilayer surface. While the residue swap does not cause any apparent negative effects on the protein structure in the variant simulations, it could adversely affect the SynGAP-membrane association in reality. The positively charged Arg329 side chain forms hydrogen bonds with other loop residues (e.g., Ser371, Asp338) that are expected to dynamically interact with the membrane head group region. However, this phenomenon is beyond the scope of the solvent-only simulations to unravel. Notably, histidine can also be double protonated and positively charged, but this alternative protonation state was not considered in the variant simulations.
c.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.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.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.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.2060G>A	R687Q (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1				-10.002	Likely Pathogenic	0.575	Likely Pathogenic	Likely Benign	0.401	Likely Benign	0.92	Ambiguous	0.1	-0.37	Likely Benign	0.28	Likely Benign	1.55	Destabilizing	-3.37	Deleterious	1.000	Probably Damaging	0.844	Possibly Damaging	3.91	Benign	0.03	Affected	3.42	17	1	1	1.0	-28.06
c.2075T>A	L692Q (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-13.873	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.596	Likely Pathogenic	3.24	Destabilizing	0.1	3.27	Destabilizing	3.26	Destabilizing	2.76	Destabilizing	-5.98	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.06	Benign	0.00	Affected	3.42	17	-2	-2	-7.3	14.97
c.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.2068T>C	S690P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.568	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.431	Likely Benign	4.84	Destabilizing	0.3	4.40	Destabilizing	4.62	Destabilizing	1.42	Destabilizing	-4.77	Deleterious	0.998	Probably Damaging	0.790	Possibly Damaging	3.44	Benign	0.01	Affected	3.42	17	1	-1	-0.8	10.04	207.5	15.1	0.1	0.0	-0.1	0.2	X	X						Potentially Pathogenic	The hydroxyl side chain of Ser690, located in an α-helix (res. Leu696-Leu685), forms a hydrogen bond with the backbone carbonyl group of Ser410 in an anti-parallel β-sheet of the C2 domain (res. Ile411-Ala399). In the variant simulations, the pyrrolidine side chain of Pro690 cannot form hydrogen bonds with the C2 domain residue, resulting in the loss of this inter-domain connection. Additionally, prolines lack a free amide group necessary for hydrogen bonding with the carbonyl group of Gly686, introducing a slight bend in the α-helix and compromising its integrity.
c.2075T>C	L692P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.447	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.668	Likely Pathogenic	9.19	Destabilizing	0.1	13.20	Destabilizing	11.20	Destabilizing	1.69	Destabilizing	-6.98	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	3.06	Benign	0.00	Affected	3.42	17	-3	-3	-5.4	-16.04	186.2	62.8	-0.2	0.1	-0.7	0.3	X							Potentially Pathogenic	The isobutyl side chain of Leu692, located in the middle of an α-helix (res. Leu685-Gln702), engages in hydrophobic packing with nearby residues (e.g., Leu441, Leu431, Leu696) in the inter-helix space. Prolines lack a free amide group necessary for hydrogen bonding with the carbonyl group of Glu688 in the same manner as Leu692 in the WT. Consequently, the residue swap with proline disrupts the continuity of the secondary structure element in the variant simulations. Additionally, the side chain of Pro692 is not as optimal as Leu692 for hydrophobic packing in the inter-helix space.
c.791T>A	L264Q (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-15.729	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.678	Likely Pathogenic	3.43	Destabilizing	0.1	2.41	Destabilizing	2.92	Destabilizing	2.48	Destabilizing	-5.52	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.49	Pathogenic	0.00	Affected	3.38	18	-2	-2	-7.3	14.97	254.7	-7.6	0.0	0.0	0.0	0.3	X		X				X	Potentially Pathogenic	The iso-butyl branched hydrocarbon side chain of Leu264, located at the end of an anti-parallel β sheet strand (res. Arg259-Arg272), packs against multiple hydrophobic residues such as Leu266, Phe314, Leu317, and Leu323 in the WT simulations. In the variant simulations, the hydrophilic carboxamide group of the Gln264 side chain is not suitable for the hydrophobic niche, causing the hydrophobic residues to make room for the swapped residue. Additionally, the carboxamide group of Gln264 forms hydrogen bonds with the backbone amide groups of Arg405 and Lys256 in the β sheet and the carbonyl group of Val350 in an α helical section of a nearby loop (res. Pro359-Phe358). The residue swap disrupts the packing of the C2 domain, which could adversely affect the C2 domain structure during folding. This disruption could potentially weaken the stability of the SynGAP-membrane association.
c.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.835C>T	R279W (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-11.417	Likely Pathogenic	0.942	Likely Pathogenic	Ambiguous	0.485	Likely Benign	2.00	Destabilizing	0.8	1.47	Ambiguous	1.74	Ambiguous	0.80	Ambiguous	-6.29	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.88	Pathogenic	0.00	Affected	3.39	18	2	-3	3.6	30.03	270.0	38.3	0.1	0.0	0.3	0.0								Uncertain	The guanidinium group of Arg279, located at the beginning of an anti-parallel β sheet strand (res. Arg279-Leu286), can form hydrogen bond with the backbone carbonyl groups of nearby loop residues (e.g., Ser296, Ser331, and As332) and form salt bridges with the carboxylate groups of Asp330 and Asp332. In the WT simulations, Arg279 sporadically forms a salt bridge even with the carboxylate group of Glu613, loosely connecting the C2 domain and GAP domain. Meanwhile, the indole ring of the Trp279 side chain is unable to hydrogen bond with the loop residues in the variant simulations. The lack of hydrogen bond or salt bridge formation with the loop residues could be significant, as Arg279 and the loops face the polar head group region of the membrane. Thus, although Trp279 could interact with the membrane surface as a “lipid anchor,” any changes to the wider loop dynamics could still adversely affect the formation of a stable SynGAP-membrane association. However, no definite conclusions on the effect of the residue swap on the SynGAP-membrane association can be drawn from solvent-only simulations.
c.844T>A	C282S (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-11.846	Likely Pathogenic	0.958	Likely Pathogenic	Likely Pathogenic	0.460	Likely Benign	1.55	Ambiguous	0.1	1.23	Ambiguous	1.39	Ambiguous	1.62	Destabilizing	-9.19	Deleterious	0.997	Probably Damaging	0.994	Probably Damaging	1.64	Pathogenic	0.03	Affected	3.39	18	0	-1	-3.3	-16.06	233.2	14.8	-0.1	0.0	-0.2	0.3						X		Potentially Benign	The thiol-containing side chain of Cys282, located at the beginning of an anti-parallel β sheet strand (res. Arg279-Leu286), packs against multiple hydrophobic residues (e.g., Ile268, Leu284, Trp308, Leu327). In the variant simulations, the hydroxyl-containing side chain of Ser282 is more hydrophilic and, hence, not as favorable as Cys282 for this hydrophobic niche. Due to this polarity difference, the residue swap could potentially weaken the hydrophobic packing of the C2 domain during the folding process.Moreover, because the C2 domain interacts with the membrane, there could also be a negative effect on the stability of the SynGAP-membrane association. However, no large-scale structural changes were observed during the variant simulations. The hydroxyl group of Ser282 forms a hydrogen bond with the backbone carbonyl group of His326 in another β strand (res. Ala322-Arg329), which competes directly with the backbone amide group of Glu283 within the secondary structure element.
c.844T>C	C282R (3D Viewer)	Likely Pathogenic	C2	Pathogenic		2				-16.378	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.466	Likely Benign	3.13	Destabilizing	0.6	1.58	Ambiguous	2.36	Destabilizing	1.70	Destabilizing	-11.03	Deleterious	0.999	Probably Damaging	0.998	Probably Damaging	1.63	Pathogenic	0.00	Affected	3.39	18	-4	-3	-7.0	53.05	297.4	-98.2	-0.1	0.1	0.5	0.0	X		X				X	Potentially Pathogenic	The thiol-containing side chain of Cys282, located at the beginning of an anti-parallel β sheet strand (res. Arg279-Leu286), is packed against multiple hydrophobic residues (e.g., Ile268, Leu284, Trp308, Leu327). In the variant simulations, the bulky side chain of Arg282 with its positively charged guanidinium group is not suitable for this hydrophobic niche. Consequently, the hydrophobic residues must either make room to accommodate Arg282 or it must escape the hydrophobic C2 domain core.As a result, new hydrogen bonds are formed with the backbone carbonyl groups of the surrounding β sheet residues Ala399, Leu325, and His326, which decreases the unity of the secondary structure elements. Notably, it is likely that the residue swap causes major problems during the C2 domain folding that are not visible in the variant simulations. In fact, even increased lability in the C2 domain could adversely affect the establishment of a stable SynGAP-membrane association.
c.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.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.812C>A	A271D (3D Viewer)	Likely Pathogenic	C2	Pathogenic		1				-18.590	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.706	Likely Pathogenic	4.71	Destabilizing	0.4	2.67	Destabilizing	3.69	Destabilizing	1.59	Destabilizing	-5.52	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.62	Pathogenic	0.00	Affected	3.38	19	0	-2	-5.3	44.01	226.2	-63.4	0.0	0.0	0.9	0.1	X		X	X			X	Potentially Pathogenic	The methyl group of Ala271, located near the end of an anti-parallel β sheet strand (res. Arg259-Arg272), packs against multiple hydrophobic residues such as Val400, Val306, and Leu274 in the WT simulations. In the variant simulations, the carboxylate group of Asp271 is not suitable for the hydrophobic niche, causing the hydrophobic residues to make room for the swapped residue. Additionally, the carboxylate group of the Asp271 side chain forms hydrogen bonds with the backbone amide groups of Arg272 and Ala399 in the β sheet, or even forms a salt bridge with the amino group of the Lys394 side chain. This directly affects the integrity of the anti-parallel β sheet at the end. In short, the residue swap disrupts the C2 domain packing during folding, which could weaken the stability of the SynGAP-membrane association.
c.815G>A	R272Q (3D Viewer)		C2	Uncertain		2	6-33437720-G-A	14	8.67e-6	-9.559	Likely Pathogenic	0.286	Likely Benign	Likely Benign	0.321	Likely Benign	0.73	Ambiguous	0.1	0.15	Likely Benign	0.44	Likely Benign	1.00	Destabilizing	-1.81	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	1.88	Pathogenic	0.03	Affected	3.38	19	1	1	1.0	-28.06	255.7	52.9	0.0	0.0	-0.2	0.1				X				Uncertain	The guanidinium group of Arg272, located at the end of an anti-parallel β sheet strand (res. Arg259-Arg272), is stably maintained in an upright and outward position via stacking with the indole ring of the Trp362 side chain in another β strand (res. Thr359-Pro364). In the WT simulations, Arg272 forms hydrogen bonds with the glycine-rich Ω loop residues (res. Val365-Pro398, e.g., Gly380) and creates a salt bridge with the carboxylate group of the Asp304 side chain.In the variant simulations, the carboxamide group of the Gln272 side chain does not stack with the indole ring of Trp362 as stably as the guanidinium group of Arg272 in the WT. Consequently, the Gln272 side chain is freer to interact with the loop residues than Arg272, potentially negatively affecting the dynamic SynGAP-membrane association. Additionally, Arg272 faces the RasGTPase interface, so the residue swap could impact the SynGAP-Ras complex formation and GTPase activation.
c.821T>A	L274Q (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-15.518	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.774	Likely Pathogenic	2.54	Destabilizing	0.3	1.74	Ambiguous	2.14	Destabilizing	1.97	Destabilizing	-5.42	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.00	Pathogenic	0.00	Affected	3.38	19	-2	-2	-7.3	14.97	245.9	1.8	0.0	0.0	0.1	0.2	X		X				X	Potentially Pathogenic	The aliphatic side chain of Leu274, located in a β hairpin loop (res. Glu273-Lys278) connecting two anti-parallel β sheet strands, packs against multiple hydrophobic residues facing the β sheet (e.g., Ala271, Leu327, Tyr280, Val306). The hydrophilic carboxamide group of the Gln274 side chain is not suitable for this hydrophobic niche, causing nearby residues to adjust to make room for the hydrophilic glutamine. Additionally, a new hydrogen bond forms with the backbone carboxyl group of Arg272 in another β strand (res. Glu273-Arg259).As a result, the backbone amide group of Ala399 and the carbonyl group of Arg272, which connect two β strands at the β sheet end, form fewer hydrogen bonds in the variant than in the WT simulations. Although no major secondary structure disruption is observed in the variant simulations, the residue swap could profoundly affect the C2 domain folding, as the hydrophobic packing of Leu274 is crucial for maintaining the loop's contact with the rest of the C2 domain. Lastly, because the Leu274-containing loop faces the membrane surface, the residue swap could also negatively impact the SynGAP-membrane association.
c.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.896G>A	R299H (3D Viewer)		C2	Conflicting		2	6-33437801-G-A	10	6.20e-6	-7.731	In-Between	0.388	Ambiguous	Likely Benign	0.238	Likely Benign	3.97	Destabilizing	1.0	0.94	Ambiguous	2.46	Destabilizing	1.41	Destabilizing	-3.35	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.69	Pathogenic	0.02	Affected	3.39	19	2	0	1.3	-19.05	211.2	72.5	-0.1	0.2	-0.2	0.3	X							Potentially Pathogenic	The guanidinium group of Arg299, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Met289-Pro298, res. Thr305-Asn315), forms hydrogen bonds that stabilize the tight turn. In the WT simulations, the Arg299 side chain hydrogen bonds with the loop backbone carbonyl groups (e.g., Ser302, Thr305, Leu274, Gly303), the hydroxyl group of Ser300, and even forms a salt bridge with the carboxylate group of Asp304.In the variant simulations, the imidazole ring of His299 (epsilon protonated state) hydrogen bonds with the carbonyl group of Asp304 and the hydroxyl group of Ser300. However, it does not form as many or as strong interactions as arginine, which could affect the initial formation of the secondary hairpin loop during folding. β hairpins are potential nucleation sites during the initial stages of protein folding, so even minor changes in them could be significant.Additionally, His299 prefers to hydrophobically interact with other hydrophobic residues inside the C2 domain core (e.g., Val306, Leu274), which destabilizes the C2 domain. Indeed, the β strand partially unfolds during the second simulation. Moreover, the positively charged Arg299 side chain faces the polar head group region of the inner leaflet membrane and could directly anchor the C2 domain to the membrane. In short, the residue swap could negatively affect both protein folding and the stability of the SynGAP-membrane association.
c.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.917T>A	V306D (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-18.289	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.530	Likely Pathogenic	4.40	Destabilizing	0.3	4.29	Destabilizing	4.35	Destabilizing	2.44	Destabilizing	-5.44	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.74	Pathogenic	0.00	Affected	3.38	19	-2	-3	-7.7	15.96	212.3	-18.3	-0.2	0.4	0.0	0.2	X		X				X	Potentially Pathogenic	The isopropyl group of Val396, located at the beginning of an anti-parallel β sheet strand (res. Thr305-Asn315), packs against multiple hydrophobic residues (e.g., Leu274, Trp308, Ala271) in the WT simulations. However, in the variant simulations, the negatively charged carboxylate group of the Asp306 side chain is not suitable for this hydrophobic niche. Consequently, the side chain moves out to interact with Ser300 in the β strand (res. Met289-Arg299) and the guanidinium group of Arg299 in the β hairpin loop.In the third simulation, the residue swap disrupts the C2 domain secondary structure and tertiary assembly to a large degree when the amino group of the Lys297 side chain rotates to form a salt bridge with Asp306. This drastic effect could potentially reflect the challenge presented by the residue swap during the C2 domain folding. Because the residue swap affects the C2 domain structure, the SynGAP-membrane association could also be impacted. However, this is beyond the scope of the solvent-only simulations to unravel.
c.922T>C	W308R (3D Viewer)	Likely Pathogenic	C2	Pathogenic		1				-12.264	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.868	Likely Pathogenic	5.40	Destabilizing	0.5	4.27	Destabilizing	4.84	Destabilizing	1.88	Destabilizing	-12.87	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.48	Pathogenic	0.00	Affected	3.38	19	2	-3	-3.6	-30.03	290.4	-26.7	-0.1	0.1	0.0	0.2	X		X				X	Potentially Pathogenic	The indole ring of Trp308, located in an anti-parallel β sheet strand (res. Thr305-Asn315), packs against multiple hydrophobic residues (e.g., Ile268, Val306, Cys282). The indole group of Trp308 also hydrogen bonds with the backbone atoms of the C2 domain residues forming the anti-parallel β sheet (e.g., Tyr280, Thr294). The guanidinium group of Arg308 is comparably sized to the tryptophan it replaced; however, it is also positively charged.In the variant simulations, the charged side chain remains buried deep in the hydrophobic part of the C2 domain, where it forms new hydrogen bonds with the backbone carbonyl atoms of surrounding residues (e.g., Val306, Ile268). However, the residue swap is likely to disrupt the hydrophobic packing during folding. At a minimum, the residue swap could affect the C2 domain stability and membrane association.
c.924G>C	W308C (3D Viewer)	Likely Pathogenic	C2	Pathogenic/Likely path.		2				-12.791	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.738	Likely Pathogenic	5.56	Destabilizing	0.3	4.38	Destabilizing	4.97	Destabilizing	1.26	Destabilizing	-11.95	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.48	Pathogenic	0.00	Affected	3.38	19	-8	-2	3.4	-83.07	230.8	60.5	-0.3	0.1	-0.4	0.4							X	Potentially Pathogenic	The indole ring of Trp308, located in an anti-parallel β sheet strand (res. Thr305-Asn315), packs against multiple hydrophobic residues (e.g., Ile268, Val306, Cys282). The indole group of Trp308 also hydrogen bonds with the backbone atoms of the C2 domain residues forming the anti-parallel β sheet (e.g., Tyr280, Thr294). The introduced Cys308 is smaller than the tryptophan it replaced. The thiol group of the Cys308 side chain is well-suited for the inner hydrophobic part of the C2 domain. Although the negative effects are essentially missing from the simulations, the side chain size difference between the residues is likely to disrupt the hydrophobic packing during folding. At a minimum, the residue swap could affect the C2 domain stability and membrane association.
c.928G>A	E310K (3D Viewer)	Likely Pathogenic	C2	Conflicting		4				-14.601	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.764	Likely Pathogenic	1.97	Ambiguous	1.2	3.66	Destabilizing	2.82	Destabilizing	1.02	Destabilizing	-3.68	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	1.19	Pathogenic	0.01	Affected	3.38	19	0	1	-0.4	-0.94	213.4	58.0	0.1	0.0	0.2	0.1		X						Potentially Pathogenic	The carboxylate group of Glu310, located in an anti-parallel β sheet strand (res. Thr305-Asn315), is ideally positioned to interact with the side chain hydroxyl and backbone amide groups of Thr295 on a twisted anti-parallel β strand (res. Met289-Arg299). Because the carboxylate group can also interact with the GAP domain residues (e.g., Gln612, Tyr614), Glu310 plays a key role in maintaining the tertiary assembly between the C2 and GAP domains. In the variant simulations, the amino group of the Lys310 side chain hydrogen bonds with the GAP domain residues and forms a salt bridge with Glu613. Although no apparent negative effects are seen due to the residue swap, it is possible that the loss of hydrogen bonding with the hydroxyl group of the Thr295 side chain causes problems during folding, potentially compromising the twisting of the β sheet.
c.930G>C	E310D (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-11.218	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.666	Likely Pathogenic	1.87	Ambiguous	0.5	2.39	Destabilizing	2.13	Destabilizing	1.04	Destabilizing	-2.76	Deleterious	0.997	Probably Damaging	0.992	Probably Damaging	1.19	Pathogenic	0.02	Affected	3.38	19	3	2	0.0	-14.03	232.6	27.2	0.1	0.0	0.1	0.1						X		Potentially Benign	The carboxylate group of Glu310, located in an anti-parallel β sheet strand (res. Thr305-Asn315), is ideally positioned to interact with the hydroxyl and backbone amide groups of Thr295 on a twisted anti-parallel β strand. Because the carboxylate group can also interact with the GAP domain residues (e.g., Gln612, Tyr614), Glu310 potentially plays a key role in maintaining the tertiary assembly between the C2 and GAP domains. In the variant simulations, the carboxylate group of Asp310 can form the same interactions as glutamate; however, due to its one hydrocarbon shorter length, the connections are less stable or less optimal.
c.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.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.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.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.1067G>A	R356H (3D Viewer)	Likely Pathogenic	C2	Likely Benign		1	6-33437972-G-A	9	5.66e-6	-11.453	Likely Pathogenic	0.614	Likely Pathogenic	Likely Benign	0.314	Likely Benign	0.59	Ambiguous	0.1	-0.27	Likely Benign	0.16	Likely Benign	1.17	Destabilizing	-4.43	Deleterious	0.999	Probably Damaging	0.987	Probably Damaging	1.70	Pathogenic	0.01	Affected	3.39	22	0	2	1.3	-19.05
c.1066C>T	R356C (3D Viewer)	Likely Pathogenic	C2	Likely Benign		1	6-33437971-C-T	5	3.10e-6	-11.827	Likely Pathogenic	0.774	Likely Pathogenic	Likely Benign	0.312	Likely Benign	0.76	Ambiguous	0.0	1.19	Ambiguous	0.98	Ambiguous	0.84	Ambiguous	-7.12	Deleterious	1.000	Probably Damaging	0.990	Probably Damaging	1.67	Pathogenic	0.00	Affected	3.39	22	-4	-3	7.0	-53.05	212.3	91.0	-0.1	0.3	-0.3	0.1		X						Potentially Pathogenic	Arg356 is located in a loop that includes a short helical section and connects two anti-parallel β sheet strands (res. Gly341-Pro349, res. Thr359-Pro364). In the WT simulations, the guanidinium group of Arg356 alternately forms salt bridges with the carboxylate groups of the GAP domain residues, Glu446 and Glu698. Arg356 also forms hydrogen bonds with the hydroxyl group of the GAP domain residue Thr691 and interacts with Met409 at the C2-GAP interface.In the variant simulations, the Cys356 mutation fails to maintain any of the Arg356 interactions and only occasionally forms weak hydrogen bonds with nearby C2 domain residues (e.g., Gln407). Although no negative structural effects are observed during the simulations, Arg356 is located at the C2 and GAP domain interface, making the residue swap potentially detrimental to the tertiary structure assembly.
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.971G>A	R324Q (3D Viewer)	Likely Benign	C2	Uncertain		3	6-33437876-G-A	3	1.86e-6	-5.001	Likely Benign	0.173	Likely Benign	Likely Benign	0.307	Likely Benign	0.56	Ambiguous	0.1	0.63	Ambiguous	0.60	Ambiguous	1.02	Destabilizing	-1.17	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	1.92	Pathogenic	0.41	Tolerated	3.39	22	1	1	1.0	-28.06
c.968T>G	L323R (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-14.568	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.692	Likely Pathogenic	3.75	Destabilizing	0.4	4.47	Destabilizing	4.11	Destabilizing	2.15	Destabilizing	-4.70	Deleterious	0.999	Probably Damaging	0.969	Probably Damaging	0.59	Pathogenic	0.01	Affected	3.39	22	-3	-2	-8.3	43.03	261.8	-61.6	-0.4	0.2	0.8	0.2	X		X				X	Potentially Pathogenic	The iso-butyl side chain of Leu323, located at the beginning of an anti-parallel β sheet strand (res. Ala322-Asp330), packs against multiple hydrophobic leucine residues (e.g., Leu264, Leu266, Leu284, Leu286). In contrast, in the variant simulations, the positively charged guanidinium group of the Arg323 side chain is unsuitable for the hydrophobic niche. Consequently, the side chain either rotates away from the center of the C2 domain or, if it remains within the C2 domain core, it reorients nearby residues to form hydrogen bonds. Regardless, the residue swap extensively disrupts the C2 domain structure.
c.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.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.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.862G>A	D288N (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437767-G-A	2	1.24e-6	-10.535	Likely Pathogenic	0.521	Ambiguous	Likely Benign	0.321	Likely Benign	-0.39	Likely Benign	0.1	0.01	Likely Benign	-0.19	Likely Benign	-0.03	Likely Benign	-3.73	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.78	Pathogenic	0.05	Affected	3.38	23	1	2	0.0	-0.98
c.910G>A	D304N (3D Viewer)		C2	Uncertain		1				-6.194	Likely Benign	0.391	Ambiguous	Likely Benign	0.345	Likely Benign	0.30	Likely Benign	0.1	-0.08	Likely Benign	0.11	Likely Benign	0.21	Likely Benign	-4.18	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.81	Pathogenic	0.03	Affected	3.38	23	1	2	0.0	-0.98
c.958G>A	V320I (3D Viewer)	Likely Benign	C2	Uncertain		1				-5.220	Likely Benign	0.111	Likely Benign	Likely Benign	0.027	Likely Benign	-0.27	Likely Benign	0.2	0.66	Ambiguous	0.20	Likely Benign	0.01	Likely Benign	-0.21	Neutral	0.198	Benign	0.114	Benign	1.77	Pathogenic	0.45	Tolerated	3.38	23	3	4	0.3	14.03
c.958G>C	V320L (3D Viewer)		C2	Uncertain		1	6-33437863-G-C	6	3.72e-6	-6.207	Likely Benign	0.362	Ambiguous	Likely Benign	0.096	Likely Benign	-0.26	Likely Benign	0.2	1.33	Ambiguous	0.54	Ambiguous	0.51	Ambiguous	-1.02	Neutral	0.900	Possibly Damaging	0.373	Benign	1.78	Pathogenic	0.92	Tolerated	3.38	23	2	1	-0.4	14.03	245.8	-10.2	0.3	0.9	0.1	0.3						X		Potentially Benign	The isopropyl side chain of Val310, located in a β hairpin loop linking two anti-parallel β sheet strands (res. Thr305-Asn315, res. Ala322-Asp330), hydrophobically packs with the side chains of nearby residues (e.g., Leu286, Val350, Pro318). The hydrophobic Leu320 side chain mostly forms the same interactions; hence, the residue swap does not seem to negatively affect the protein structure based on the variant simulations.
c.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.859G>C	D287H (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-14.518	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.589	Likely Pathogenic	0.48	Likely Benign	0.3	0.32	Likely Benign	0.40	Likely Benign	0.63	Ambiguous	-6.43	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.51	Pathogenic	0.00	Affected	3.38	23	1	-1	0.3	22.05	235.6	3.8	0.1	1.2	0.1	0.1	X	X						Potentially Pathogenic	The carboxylate group of Asp287, located at the beginning of a β hairpin loop connecting two anti-parallel β sheet strands (res. Arg279-Leu286, res. Met289-Pro298), maintains a salt bridge with the guanidinium group of Arg324 in the β sheet during the WT simulations. In the variant simulations, the imidazole ring of the His287 side chain is unable to form a salt bridge with Arg324 or establish any other stable compensatory interactions, which could weaken the beta sandwich assembly of the C2 domain. This destabilization of the C2 domain could adversely affect the stability of the SynGAP-membrane association.
c.859G>T	D287Y (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-12.877	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.663	Likely Pathogenic	0.21	Likely Benign	0.2	0.48	Likely Benign	0.35	Likely Benign	0.27	Likely Benign	-8.27	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.51	Pathogenic	0.00	Affected	3.38	23	-4	-3	2.2	48.09	257.8	-44.4	-0.6	1.6	0.2	0.3	X	X						Potentially Pathogenic	The carboxylate group of Asp287, located at the beginning of a β hairpin loop linking two anti-parallel β sheet strands (res. Arg279-Leu286, res. Met289-Pro298), maintains a salt bridge with the guanidinium group of Arg324 in the β sheet during the WT simulations. In the variant simulations, the phenol group of the Tyr287 side chain is unable to form a salt bridge with the guanidinium group of Arg324, which could weaken the tertiary structure assembly of the C2 domain. However, the phenol group of Tyr287 frequently stacks with the Arg324 guanidinium side chain, which could help maintain the tertiary structure, especially compared to the D287H variant. The destabilization of the C2 domain could adversely affect the stability of the SynGAP-membrane association.
c.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.872A>G	Y291C (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-8.997	Likely Pathogenic	0.967	Likely Pathogenic	Likely Pathogenic	0.505	Likely Pathogenic	2.90	Destabilizing	0.4	3.51	Destabilizing	3.21	Destabilizing	1.35	Destabilizing	-7.37	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.76	Pathogenic	0.01	Affected	3.38	23	0	-2	3.8	-60.04	205.2	66.1	0.1	0.0	-0.4	0.4	X						X	Potentially Pathogenic	The phenol group of the Tyr291 side chain, located in an anti-parallel β sheet strand (res. Met289-Pro298), packs against hydrophobic residues of the C2 and PH domains (e.g., Leu317, Leu286, Leu284, Pro208, Val209). The phenol ring of Tyr291 also forms favorable Met-aromatic stacking with the methyl group of Met289. In the variant simulation, the thiol group of the Cys291 side chain is not as suitable for the hydrophobic inter-domain space as the phenol ring of Tyr291. Consequently, the structural unity of the PH domain is weakened and ultimately unfolds in the second simulation. Moreover, the residue swap might result in severe detrimental effects on the C2 domain structure and the C2-PH domain tertiary structure assembly during folding.
c.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.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.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.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.980T>C	L327P (3D Viewer)	Likely Pathogenic	C2	Pathogenic		3				-16.602	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.658	Likely Pathogenic	5.38	Destabilizing	0.1	4.00	Destabilizing	4.69	Destabilizing	2.62	Destabilizing	-5.97	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.52	Pathogenic	0.01	Affected	3.38	23	-3	-3	-5.4	-16.04	221.7	69.4	0.1	0.0	0.6	0.1	X							Potentially Pathogenic	The backbone amide group of Leu327, located in the middle of an anti-parallel β sheet strand (res. Ala322-Asp330), forms a hydrogen bond with the carbonyl group of Gly344 on a neighboring β strand (res. Lys336-Pro349) in the WT simulations. In contrast, in the variant simulations, the introduction of Pro327 destabilizes the hydrogen bonding between the two anti-parallel β strands because proline lacks the backbone amide group altogether. Additionally, in the WT simulations, the iso-butyl side chain of Leu327 packs against multiple hydrophobic residues (e.g., Leu274, V400, Val343), whereas the less bulky cyclic five-membered pyrrolidine ring of Pro327 cannot fill the same space as effectively. Thus, although no large-scale unfolding is observed during the variant simulations, the residue swap is likely to cause severe problems for the correct C2 domain folding, which could also affect the SynGAP-membrane association.	10.1016/j.ajhg.2020.11.011
c.1084T>C	W362R (3D Viewer)	Likely Pathogenic	C2	Pathogenic		2				-14.004	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.706	Likely Pathogenic	2.64	Destabilizing	0.3	3.90	Destabilizing	3.27	Destabilizing	1.10	Destabilizing	-12.87	Deleterious	0.999	Probably Damaging	0.996	Probably Damaging	1.28	Pathogenic	0.00	Affected	3.39	24	2	-3	-3.6	-30.03	287.5	-34.1	-0.2	0.1	-0.6	0.2	X			X			X	Potentially Pathogenic	The indole ring of Trp362, located on the surface of an anti-parallel β sheet (res. Thr359-Pro364) in the C2 domain, stacks with nearby residues (e.g., Arg401, Arg272). In the variant simulations, the guanidinium group of the introduced residue Arg362 forms a salt bridge with the carboxylate group of Glu273 and, like Trp362, stacks with other arginine residues (e.g., Arg401, Arg272). This residue is at both the C2-membrane interface and the C2-RasGTPase interface, so the residue swap could potentially affect both interactions. However, these phenomena cannot be addressed using solvent-only simulations. Notably, Arg272, which stacks with both the non-mutated Trp362 and the mutated Arg362, forms a salt bridge directly with Asp105 of Ras in the WT simulations. Therefore, the residue swap could affect the C2 domain stability, the SynGAP-membrane association, and the SynGAP-Ras association.	10.1016/j.ajhg.2020.11.011
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.1970G>T	W657L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.411	Likely Pathogenic	0.960	Likely Pathogenic	Likely Pathogenic	0.213	Likely Benign	0.14	Likely Benign	0.1	0.73	Ambiguous	0.44	Likely Benign	0.87	Ambiguous	-10.86	Deleterious	0.277	Benign	0.078	Benign	3.52	Benign	0.14	Tolerated	3.39	24	-2	-2	4.7	-73.05
c.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.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.1025A>C	Y342S (3D Viewer)	Likely Pathogenic	C2	Uncertain		2				-7.996	In-Between	0.925	Likely Pathogenic	Ambiguous	0.407	Likely Benign	3.03	Destabilizing	0.1	2.87	Destabilizing	2.95	Destabilizing	0.93	Ambiguous	-6.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.75	Pathogenic	0.04	Affected	3.37	25	-3	-2	0.5	-76.10	200.1	77.8	0.0	0.0	-0.2	0.1								Potentially Pathogenic	The phenol ring of Tyr342, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), faces outward in the C2 domain. In the WT simulations, the phenol ring of Tyr342 contributes to a triple tyrosine stack (Tyr342, Tyr328, and Tyr281) that links together three anti-parallel β sheet strands. Additionally, it shields Gly344 from the solvent, reducing its exposure and providing stability for the β-sandwich. This motif also contributes to a twist formation in the β sheet.In the variant simulations, the Ser342 side chain cannot participate in the stack formation. Instead, the hydroxyl group of the Ser342 side chain forms a hydrogen bond with the imidazole ring of His326 in a neighboring β strand (res. Ala322-Asp330). This disrupts the formation of a hydrogen bond between His326 and the carboxylate group of the Glu283 side chain from another β strand (res. Arg279-Cys285). Although these changes in surface interactions could weaken the characteristic twist that strengthens the β sheet fold, no major structural effects are observed in the variant simulations. The residue swap could also affect the SynGAP-membrane association, as the hydroxyl group of Ser342 could form hydrogen bonds with membrane-facing loop residues. However, this phenomenon cannot be addressed using solvent-only simulations.
c.1025A>G	Y342C (3D Viewer)	Likely Pathogenic	C2	Benign/Likely benign		2	6-33437930-A-G	21	1.30e-5	-7.596	In-Between	0.682	Likely Pathogenic	Likely Benign	0.404	Likely Benign	2.48	Destabilizing	0.1	2.73	Destabilizing	2.61	Destabilizing	0.92	Ambiguous	-6.67	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.72	Pathogenic	0.02	Affected	3.37	25	0	-2	3.8	-60.04	242.4	62.8	0.1	0.0	-0.1	0.2								Potentially Pathogenic	The phenol ring of Tyr342, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), faces outward in the C2 domain. This phenol ring contributes to a triple tyrosine stack (Tyr342, Tyr328, and Tyr281) that links together three anti-parallel β sheet strands. Additionally, it shields Gly344 from the solvent, reducing its exposure and providing stability for the β-sandwich. This motif also contributes to a twist formation in the β sheet.In the variant simulations, the Cys342 side chain cannot participate in the stack formation. Instead, its thiol group forms a hydrogen bond with the backbone carbonyl group of Leu327. Although these changes in surface interactions could weaken the characteristic twist that strengthens the β sheet fold, no major structural effects are observed in the variant simulations. The residue swap could also affect the SynGAP-membrane association; however, this phenomenon cannot be addressed using solvent-only simulations. Notably, the thiol group of cysteine is not a particularly strong hydrogen-bonding partner, which could mitigate the negative effects of the residue swap.
c.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.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.1058T>C	L353P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-7.913	In-Between	0.936	Likely Pathogenic	Ambiguous	0.464	Likely Benign	4.63	Destabilizing	0.1	10.19	Destabilizing	7.41	Destabilizing	2.17	Destabilizing	-3.70	Deleterious	0.947	Possibly Damaging	0.454	Possibly Damaging	1.29	Pathogenic	0.02	Affected	3.37	25	-3	-3	-5.4	-16.04
c.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.1030G>A	G344S (3D Viewer)	Likely Pathogenic	C2	Pathogenic		5				-11.254	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.790	Likely Pathogenic	9.02	Destabilizing	0.7	6.08	Destabilizing	7.55	Destabilizing	0.98	Ambiguous	-5.28	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-0.45	Pathogenic	0.04	Affected	3.37	25	1	0	-0.4	30.03	217.3	-51.7	0.0	0.1	0.2	0.1			X				X	Potentially Pathogenic	Because Gly344 lacks a proper side chain, it allows the anti-parallel β sheet strand (res. Gly341-Pro349) to have a slight twist. Within a β strand, side chains normally alternate between outward and inward positions, but glycine is an exception as it allows the alternating pattern to skip a residue. Introducing serine or any other residue with a side chain at position 344 prevents this unique skip in the alternating pattern, causing structural strain or likely preventing correct folding altogether. Additionally, Tyr342 shields Gly344 from the solvent, contributing to twist formation in the β sheet and stabilizing the β-strand.In the variant simulations, the side chain of Ser344 assumes the inward position. However, the hydrophobic niche formed by multiple C2 domain residues (e.g., Val365, Val343, Leu327) is not accommodating for its hydroxyl group. The outward position, not seen in the simulations, would be equally disadvantageous due to the presence of hydrophobic residues on that side as well (e.g., Leu345, Tyr342). Serine is also not well-suited for twist formation, as it tends to suppress twisting and bending in β sheets. At this position, the hydroxyl group of Ser344 could also form hydrogen bonds with the backbone atoms of the Gly-rich Ω loop in the C2 domain (e.g., Thr366, Leu367, Gly378; res. Pro364-Pro398), potentially adversely affecting membrane-loop dynamics and ultimately compromising the stability of the SynGAP-membrane association.
c.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.1045C>T	P349S (3D Viewer)		C2	Uncertain		1				-7.654	In-Between	0.217	Likely Benign	Likely Benign	0.277	Likely Benign	1.92	Ambiguous	0.1	2.28	Destabilizing	2.10	Destabilizing	0.87	Ambiguous	-6.13	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	1.66	Pathogenic	0.06	Tolerated	3.37	25	1	-1	0.8	-10.04	194.9	-18.1	-0.1	0.0	0.2	0.1	X						X	Potentially Pathogenic	The cyclic pyrrolidine side chain of Pro349, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), allows the strand to end and make a tight turn before a short α helical section within a loop connecting to another β strand (res. Thr359-Pro364). In the variant simulations, the hydroxyl group of Ser349 forms a hydrogen bond with the backbone amide group of Ala351 in the short helical section. Conversely, the backbone amide group of Ser349 (absent in proline) does not form any intra-protein hydrogen bonds. However, the β strand end connects to the α helical section in a more stable and consistent manner compared to the WT. Although the residue swap does not cause major adverse effects on the protein structure in the simulations, it is possible that the tight turn at the β strand end could not be created during folding without the presence of proline.