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.3308G>T	R1103L	Likely Benign		Uncertain		1	6-33443860-G-T			-2.330	Likely Benign	0.205	Likely Benign	Likely Benign	0.173	Likely Benign										-2.35	Neutral	0.002	Benign	0.005	Benign	2.44	Pathogenic	0.02	Affected	3.77	5	-3	-2	8.3	-43.03
c.3313C>T	R1105W			Uncertain		1	6-33443865-C-T	6	3.93e-6	-6.911	Likely Benign	0.488	Ambiguous	Likely Benign	0.133	Likely Benign										-4.34	Deleterious	0.999	Probably Damaging	0.696	Possibly Damaging	2.42	Pathogenic	0.02	Affected	3.77	5	-3	2	3.6	30.03
c.3314G>A	R1105Q	Likely Benign		Uncertain		2	6-33443866-G-A	3	1.96e-6	-3.666	Likely Benign	0.216	Likely Benign	Likely Benign	0.104	Likely Benign										-1.21	Neutral	0.958	Probably Damaging	0.194	Benign	2.50	Benign	0.16	Tolerated	3.77	5	1	1	1.0	-28.06
c.3323G>T	S1108I			Uncertain		1	6-33443875-G-T			-3.666	Likely Benign	0.292	Likely Benign	Likely Benign	0.145	Likely Benign										-3.73	Deleterious	0.971	Probably Damaging	0.604	Possibly Damaging	2.44	Pathogenic	0.10	Tolerated	3.77	5	-2	-1	5.3	26.08
c.3338G>A	G1113D	Likely Benign		Uncertain		1	6-33443890-G-A			-4.638	Likely Benign	0.354	Ambiguous	Likely Benign	0.061	Likely Benign										-0.72	Neutral	0.029	Benign	0.017	Benign	2.58	Benign	0.34	Tolerated	4.32	2	-1	1	-3.1	58.04
c.3354C>A	S1118R	Likely Benign		Uncertain		1				-2.670	Likely Benign	0.553	Ambiguous	Likely Benign	0.166	Likely Benign										-0.74	Neutral	0.034	Benign	0.023	Benign	5.17	Benign	0.05	Affected	4.32	2	-1	0	-3.7	69.11
c.335G>C	G112A	Likely Benign		Uncertain		1	6-33432200-G-C	15	9.30e-6	-2.456	Likely Benign	0.119	Likely Benign	Likely Benign	0.114	Likely Benign										-2.34	Neutral	0.231	Benign	0.054	Benign	4.07	Benign	0.00	Affected	3.61	5	1	0	2.2	14.03
c.3361A>G	S1121G	Likely Benign		Uncertain		1	6-33443913-A-G	1	7.00e-7	-1.220	Likely Benign	0.054	Likely Benign	Likely Benign	0.067	Likely Benign										-0.53	Neutral	0.003	Benign	0.004	Benign	6.63	Benign	0.00	Affected	3.77	5	0	1	0.4	-30.03
c.3368G>A	G1123D			Uncertain		1	6-33443920-G-A	2	1.33e-6	-10.321	Likely Pathogenic	0.405	Ambiguous	Likely Benign	0.360	Likely Benign										-0.78	Neutral	0.500	Possibly Damaging	0.157	Benign	4.34	Benign	0.19	Tolerated	3.77	5	1	-1	-3.1	58.04
c.3374G>C	G1125A	Likely Benign		Uncertain		1	6-33443926-G-C	1	6.68e-7	-6.569	Likely Benign	0.083	Likely Benign	Likely Benign	0.232	Likely Benign										-0.60	Neutral	0.999	Probably Damaging	0.995	Probably Damaging	4.60	Benign	0.11	Tolerated	3.77	5	1	0	2.2	14.03
c.3376G>T	G1126C	Likely Benign		Uncertain		1	6-33443928-G-T	11	7.35e-6	-9.389	Likely Pathogenic	0.113	Likely Benign	Likely Benign	0.449	Likely Benign										-1.40	Neutral	0.005	Benign	0.005	Benign	4.74	Benign	0.02	Affected	3.77	5	-3	-3	2.9	46.09
c.3377G>A	G1126D			Uncertain		1				-8.888	Likely Pathogenic	0.432	Ambiguous	Likely Benign	0.376	Likely Benign										-0.65	Neutral	0.906	Possibly Damaging	0.473	Possibly Damaging	4.82	Benign	0.02	Affected	3.77	5	1	-1	-3.1	58.04
c.3377G>T	G1126V	Likely Benign		Uncertain		1	6-33443929-G-T			-6.536	Likely Benign	0.089	Likely Benign	Likely Benign	0.357	Likely Benign										-1.20	Neutral	0.009	Benign	0.008	Benign	4.76	Benign	0.03	Affected	3.77	5	-1	-3	4.6	42.08
c.3379G>A	G1127R	Likely Benign		Uncertain		1	6-33443931-G-A	2	1.34e-6	-5.949	Likely Benign	0.629	Likely Pathogenic	Likely Benign	0.341	Likely Benign										-0.87	Neutral	0.001	Benign	0.001	Benign	4.86	Benign	0.12	Tolerated	4.32	4	-2	-3	-4.1	99.14
c.3380G>T	G1127V	Likely Benign		Uncertain		1	6-33443932-G-T	1	6.69e-7	-6.097	Likely Benign	0.094	Likely Benign	Likely Benign	0.230	Likely Benign										-1.01	Neutral	0.004	Benign	0.005	Benign	4.81	Benign	0.17	Tolerated	4.32	4	-1	-3	4.6	42.08
c.3386T>C	L1129P	Likely Benign		Uncertain		2				-2.991	Likely Benign	0.154	Likely Benign	Likely Benign	0.432	Likely Benign										0.27	Neutral	0.971	Probably Damaging	0.773	Possibly Damaging	5.44	Benign	0.00	Affected	4.32	4	-3	-3	-5.4	-16.04
c.3405G>C	K1135N	Likely Benign		Uncertain		1				-5.715	Likely Benign	0.960	Likely Pathogenic	Likely Pathogenic	0.166	Likely Benign										-0.97	Neutral	0.411	Benign	0.321	Benign	5.43	Benign	0.07	Tolerated	4.32	2	1	0	0.4	-14.07
c.3413C>A	S1138Y			Uncertain		1	6-33444448-C-A	3	1.86e-6	-6.610	Likely Benign	0.449	Ambiguous	Likely Benign	0.391	Likely Benign										-2.51	Deleterious	0.997	Probably Damaging	0.996	Probably Damaging	5.41	Benign	0.05	Affected	4.32	4	-2	-3	-0.5	76.10
c.3434A>G	N1145S	Likely Benign		Uncertain		1	6-33444469-A-G	2	1.24e-6	-0.989	Likely Benign	0.126	Likely Benign	Likely Benign	0.308	Likely Benign										-1.15	Neutral	0.997	Probably Damaging	0.989	Probably Damaging	5.55	Benign	0.89	Tolerated	4.32	4	1	1	2.7	-27.03
c.3442A>T	M1148L	Likely Benign		Uncertain		1				-1.777	Likely Benign	0.093	Likely Benign	Likely Benign	0.068	Likely Benign										-1.13	Neutral	0.016	Benign	0.016	Benign	2.62	Benign	0.00	Affected	4.32	2	4	2	1.9	-18.03
c.3449C>T	A1150V	Likely Benign		Uncertain		1	6-33444484-C-T	3	1.86e-6	-3.648	Likely Benign	0.192	Likely Benign	Likely Benign	0.066	Likely Benign										-2.22	Neutral	0.114	Benign	0.055	Benign	2.32	Pathogenic	0.04	Affected	3.77	5	0	0	2.4	28.05
c.3457C>T	R1153W	Likely Pathogenic		Uncertain		2	6-33444492-C-T	2	1.24e-6	-5.812	Likely Benign	0.994	Likely Pathogenic	Likely Pathogenic	0.317	Likely Benign										-5.88	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.46	Pathogenic	0.00	Affected	3.77	5	2	-3	3.6	30.03
c.3484C>T	P1162S	Likely Benign		Uncertain		1				-2.118	Likely Benign	0.913	Likely Pathogenic	Ambiguous	0.215	Likely Benign										-1.93	Neutral	1.000	Probably Damaging	0.999	Probably Damaging	2.73	Benign	0.55	Tolerated	3.88	3	1	-1	0.8	-10.04
c.3487C>G	H1163D			Uncertain		1				-2.107	Likely Benign	0.949	Likely Pathogenic	Ambiguous	0.476	Likely Benign										-2.60	Deleterious	0.991	Probably Damaging	0.991	Probably Damaging	5.44	Benign	0.31	Tolerated	3.88	3	1	-1	-0.3	-22.05
c.3502A>G	I1168V	Likely Benign		Uncertain		1				-3.263	Likely Benign	0.524	Ambiguous	Likely Benign	0.363	Likely Benign										-0.14	Neutral	0.876	Possibly Damaging	0.643	Possibly Damaging	5.47	Benign	0.84	Tolerated	3.88	3	4	3	-0.3	-14.03
c.3508A>G	S1170G	Likely Benign	Coiled-coil	Uncertain		1				-4.288	Likely Benign	0.221	Likely Benign	Likely Benign	0.349	Likely Benign										-0.81	Neutral	0.241	Benign	0.229	Benign	5.31	Benign	0.54	Tolerated	4.32	4	1	0	0.4	-30.03
c.3511G>A	A1171T	Likely Benign	Coiled-coil	Uncertain		1				-3.658	Likely Benign	0.149	Likely Benign	Likely Benign	0.201	Likely Benign										-0.48	Neutral	0.245	Benign	0.138	Benign	5.45	Benign	0.07	Tolerated	4.32	4	1	0	-2.5	30.03
c.3511_3512delinsTG	A1171C	Likely Benign	Coiled-coil	Uncertain		1				-5.363	Likely Benign	0.496	Ambiguous	Likely Benign												-1.16	Neutral	0.978	Probably Damaging	0.825	Possibly Damaging	5.32	Benign	0.02	Affected	4.32	4	-2	0	0.7	32.06
c.3520G>A	E1174K	Likely Benign	Coiled-coil	Uncertain		1	6-33444555-G-A	2	1.24e-6	-4.345	Likely Benign	0.898	Likely Pathogenic	Ambiguous	0.442	Likely Benign										-1.59	Neutral	0.962	Probably Damaging	0.367	Benign	5.52	Benign	0.03	Affected	4.32	2	0	1	-0.4	-0.94
c.3529G>A	E1177K	Likely Benign	Coiled-coil	Uncertain		1				-3.413	Likely Benign	0.944	Likely Pathogenic	Ambiguous	0.560	Likely Pathogenic										-1.75	Neutral	0.905	Possibly Damaging	0.637	Possibly Damaging	5.44	Benign	0.11	Tolerated	4.32	2	0	1	-0.4	-0.94
c.3557C>T	S1186L		Coiled-coil	Uncertain		1	6-33444592-C-T			-4.829	Likely Benign	0.923	Likely Pathogenic	Ambiguous	0.177	Likely Benign										-2.58	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	2.65	Benign	0.04	Affected	3.82	4	-3	-2	4.6	26.08
c.3572G>A	R1191Q	Likely Benign	Coiled-coil	Uncertain		2	6-33444607-G-A	9	5.58e-6	-1.069	Likely Benign	0.943	Likely Pathogenic	Ambiguous	0.343	Likely Benign										-1.41	Neutral	0.998	Probably Damaging	0.992	Probably Damaging	2.68	Benign	0.08	Tolerated	3.82	4	1	1	1.0	-28.06
c.3574C>G	L1192V	Likely Benign	Coiled-coil	Uncertain		1				-4.132	Likely Benign	0.471	Ambiguous	Likely Benign	0.041	Likely Benign										-0.89	Neutral	0.779	Possibly Damaging	0.527	Possibly Damaging	2.69	Benign	0.16	Tolerated			2	1	0.4	-14.03
c.3595G>A	E1199K		Coiled-coil	Uncertain		1	6-33446587-G-A	1	6.20e-7	-10.853	Likely Pathogenic	0.954	Likely Pathogenic	Ambiguous	0.171	Likely Benign										-2.26	Neutral	1.000	Probably Damaging	0.995	Probably Damaging	2.52	Benign	0.00	Affected	3.77	5	0	1	-0.4	-0.94
c.3607C>G	H1203D	Likely Benign	Coiled-coil	Uncertain		1				-6.729	Likely Benign	0.525	Ambiguous	Likely Benign	0.403	Likely Benign										-1.89	Neutral	0.473	Possibly Damaging	0.265	Benign	5.51	Benign	0.24	Tolerated	3.77	5	1	-1	-0.3	-22.05
c.3607C>T	H1203Y	Likely Benign	Coiled-coil	Uncertain		1	6-33446599-C-T	2	1.24e-6	-6.834	Likely Benign	0.149	Likely Benign	Likely Benign	0.233	Likely Benign										-1.52	Neutral	0.006	Benign	0.011	Benign	5.55	Benign	0.10	Tolerated	3.77	5	2	0	1.9	26.03
c.3614T>C	L1205P	Likely Pathogenic	Coiled-coil	Uncertain		1				-16.878	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.536	Likely Pathogenic										-5.91	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.45	Pathogenic	0.00	Affected			-3	-3	-5.4	-16.04
c.3633G>A	M1211I	Likely Benign	Coiled-coil	Uncertain		1	6-33446625-G-A	3	1.86e-6	-1.537	Likely Benign	0.764	Likely Pathogenic	Likely Benign	0.298	Likely Benign										-0.42	Neutral	0.969	Probably Damaging	0.968	Probably Damaging	5.40	Benign	1.00	Tolerated	3.77	5	1	2	2.6	-18.03
c.3640C>T	R1214W	Likely Pathogenic	Coiled-coil	Uncertain		1	6-33446632-C-T	2	1.24e-6	-8.799	Likely Pathogenic	0.710	Likely Pathogenic	Likely Benign	0.143	Likely Benign										-4.95	Deleterious	1.000	Probably Damaging	0.983	Probably Damaging	2.45	Pathogenic	0.00	Affected	3.77	5	2	-3	3.6	30.03
c.3653A>T	E1218V	Likely Pathogenic	Coiled-coil	Uncertain		2				-5.647	Likely Benign	0.936	Likely Pathogenic	Ambiguous	0.418	Likely Benign										-5.68	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.21	Pathogenic	0.00	Affected	3.77	5	-2	-2	7.7	-29.98
c.3655T>C	Y1219H	Likely Pathogenic	Coiled-coil	Uncertain		1				-9.511	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.363	Likely Benign										-3.62	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.15	Pathogenic	0.00	Affected	3.77	5	0	2	-1.9	-26.03
c.3686A>C	Q1229P	Likely Pathogenic	Coiled-coil	Uncertain		1				-10.397	Likely Pathogenic	0.980	Likely Pathogenic	Likely Pathogenic	0.422	Likely Benign										-3.69	Deleterious	0.998	Probably Damaging	0.995	Probably Damaging	1.75	Pathogenic	0.12	Tolerated	3.77	5	0	-1	1.9	-31.01
c.36C>G	S12R	Likely Benign		Uncertain		1	6-33420300-C-G	4	2.59e-6	-4.033	Likely Benign	0.500	Ambiguous	Likely Benign	0.097	Likely Benign										-0.30	Neutral	0.000	Benign	0.000	Benign	4.09	Benign	0.00	Affected	4.32	1	0	-1	-3.7	69.11
c.3705G>A	M1235I	Likely Benign	Coiled-coil	Uncertain		1				-4.312	Likely Benign	0.310	Likely Benign	Likely Benign	0.027	Likely Benign										-1.44	Neutral	0.139	Benign	0.056	Benign	2.69	Benign	0.04	Affected	3.77	5	1	2	2.6	-18.03
c.3721C>A	L1241M		Coiled-coil	Uncertain		1				-5.881	Likely Benign	0.782	Likely Pathogenic	Likely Benign	0.167	Likely Benign										-1.43	Neutral	1.000	Probably Damaging	0.999	Probably Damaging	1.65	Pathogenic	0.00	Affected			4	2	-1.9	18.03
c.3731G>A	S1244N	Likely Pathogenic	Coiled-coil	Uncertain		1				-9.008	Likely Pathogenic	0.751	Likely Pathogenic	Likely Benign	0.154	Likely Benign										-1.87	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.10	Pathogenic	0.15	Tolerated	3.77	5	1	1	-2.7	27.03
c.373C>T	P125S	Likely Benign		Uncertain		1				-3.769	Likely Benign	0.238	Likely Benign	Likely Benign	0.121	Likely Benign										-3.57	Deleterious	0.580	Possibly Damaging	0.140	Benign	2.86	Benign	0.02	Affected	3.61	5	1	-1	0.8	-10.04
c.3773A>G	Q1258R	Likely Pathogenic	Coiled-coil	Uncertain		1				-10.971	Likely Pathogenic	0.931	Likely Pathogenic	Ambiguous	0.316	Likely Benign										-3.19	Deleterious	0.994	Probably Damaging	0.988	Probably Damaging	2.00	Pathogenic	0.00	Affected			1	1	-1.0	28.06
c.3788T>C	I1263T	Likely Pathogenic	Coiled-coil	Uncertain		1	6-33446780-T-C	2	1.24e-6	-6.564	Likely Benign	0.962	Likely Pathogenic	Likely Pathogenic	0.529	Likely Pathogenic										-4.15	Deleterious	0.946	Possibly Damaging	0.673	Possibly Damaging	1.81	Pathogenic	0.00	Affected	3.77	5	0	-1	-5.2	-12.05
c.379C>T	R127W			Uncertain		1				-4.776	Likely Benign	0.806	Likely Pathogenic	Ambiguous	0.118	Likely Benign										-2.98	Deleterious	0.989	Probably Damaging	0.420	Benign	3.88	Benign	0.00	Affected			2	-3	3.6	30.03
c.37A>G	I13V	Likely Benign		Uncertain		1				-2.497	Likely Benign	0.105	Likely Benign	Likely Benign	0.110	Likely Benign										0.01	Neutral	0.000	Benign	0.000	Benign	4.25	Benign	0.00	Affected			4	3	-0.3	-14.03
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.380G>A	R127Q	Likely Benign		Uncertain		1	6-33432245-G-A	6	3.72e-6	-1.711	Likely Benign	0.320	Likely Benign	Likely Benign	0.037	Likely Benign										-1.04	Neutral	0.006	Benign	0.001	Benign	4.04	Benign	0.02	Affected	3.74	4	1	1	1.0	-28.06
c.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.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.382C>A	P128T	Likely Benign		Uncertain		1	6-33432247-C-A	1	6.20e-7	-4.217	Likely Benign	0.267	Likely Benign	Likely Benign	0.075	Likely Benign										-0.96	Neutral	0.952	Possibly Damaging	0.500	Possibly Damaging	4.19	Benign	0.35	Tolerated	3.74	4	-1	0	0.9	3.99
c.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.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.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>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.3906G>C	L1302F			Uncertain		1				-5.674	Likely Benign	0.148	Likely Benign	Likely Benign	0.211	Likely Benign										-2.70	Deleterious	0.960	Probably Damaging	0.657	Possibly Damaging	1.53	Pathogenic	0.00	Affected			2	0	-1.0	34.02
c.3907G>A	G1303S	Likely Benign		Uncertain		1				-2.271	Likely Benign	0.125	Likely Benign	Likely Benign	0.155	Likely Benign										-0.19	Neutral	0.649	Possibly Damaging	0.433	Benign	2.84	Benign	0.18	Tolerated			1	0	-0.4	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.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.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.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.3974C>T	P1325L	Likely Benign		Uncertain		1	6-33451848-C-T			-5.256	Likely Benign	0.085	Likely Benign	Likely Benign	0.146	Likely Benign										-1.05	Neutral	0.000	Benign	0.000	Benign	4.05	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
c.3977C>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.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.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>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>C	R136P	Likely Pathogenic		Uncertain		1				-11.952	Likely Pathogenic	0.981	Likely Pathogenic	Likely Pathogenic	0.277	Likely Benign										-3.72	Deleterious	0.910	Possibly Damaging	0.578	Possibly Damaging	3.47	Benign	0.00	Affected	3.61	5	0	-2	2.9	-59.07
c.416G>A	S139N	Likely Benign		Uncertain		1	6-33432713-G-A	3	2.22e-6	-4.584	Likely Benign	0.688	Likely Pathogenic	Likely Benign	0.109	Likely Benign										-0.75	Neutral	0.149	Benign	0.047	Benign	4.14	Benign	0.24	Tolerated	3.61	5	1	1	-2.7	27.03
c.431C>T	T144M	Likely Pathogenic		Uncertain		2	6-33432728-C-T	2	1.30e-6	-11.228	Likely Pathogenic	0.922	Likely Pathogenic	Ambiguous	0.118	Likely Benign										-3.16	Deleterious	0.913	Possibly Damaging	0.333	Benign	3.73	Benign	0.00	Affected	3.61	5	-1	-1	2.6	30.09
c.43G>A	A15T	Likely Benign		Uncertain		1	6-33420307-G-A	4	2.60e-6	-3.720	Likely Benign	0.125	Likely Benign	Likely Benign	0.086	Likely Benign										-0.08	Neutral	0.602	Possibly Damaging	0.017	Benign	4.16	Benign	0.00	Affected	4.32	1	1	0	-2.5	30.03
c.43G>C	A15P	Likely Benign		Uncertain		1				-3.436	Likely Benign	0.097	Likely Benign	Likely Benign	0.146	Likely Benign										-0.23	Neutral	0.880	Possibly Damaging	0.123	Benign	4.09	Benign	0.00	Affected			1	-1	-3.4	26.04
c.44C>T	A15V	Likely Benign		Uncertain		1	6-33420308-C-T	1	6.49e-7	-3.560	Likely Benign	0.161	Likely Benign	Likely Benign	0.105	Likely Benign										0.20	Neutral	0.602	Possibly Damaging	0.015	Benign	4.19	Benign	0.00	Affected	4.32	1	0	0	2.4	28.05
c.451G>C	D151H	Likely Pathogenic		Uncertain		1	6-33432748-G-C	2	1.26e-6	-11.747	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.335	Likely Benign										-3.90	Deleterious	0.999	Probably Damaging	0.995	Probably Damaging	3.86	Benign	0.00	Affected	3.61	5	-1	1	0.3	22.05
c.453C>A	D151E	Likely Benign		Uncertain		1				-5.662	Likely Benign	0.886	Likely Pathogenic	Ambiguous	0.142	Likely Benign										-2.02	Neutral	0.984	Probably Damaging	0.967	Probably Damaging	3.99	Benign	0.11	Tolerated	3.61	5	3	2	0.0	14.03
c.455G>A	R152Q			Uncertain		1	6-33432752-G-A	5	3.14e-6	-10.336	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.181	Likely Benign										-2.34	Neutral	0.997	Probably Damaging	0.968	Probably Damaging	3.89	Benign	0.00	Affected	3.61	5	1	1	1.0	-28.06
c.467T>G	F156C	Likely Pathogenic		Uncertain		1				-13.658	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.297	Likely Benign										-3.54	Deleterious	0.999	Probably Damaging	0.990	Probably Damaging	3.92	Benign	0.00	Affected			-4	-2	-0.3	-44.04
c.470G>A	R157H			Uncertain		1	6-33432767-G-A	1	6.20e-7	-10.235	Likely Pathogenic	0.604	Likely Pathogenic	Likely Benign	0.254	Likely Benign										-2.23	Neutral	0.999	Probably Damaging	0.987	Probably Damaging	3.80	Benign	0.00	Affected	3.74	4	2	0	1.3	-19.05
c.484C>G	R162G	Likely Benign		Uncertain		1				-6.985	Likely Benign	0.664	Likely Pathogenic	Likely Benign	0.190	Likely Benign										-0.73	Neutral	0.487	Possibly Damaging	0.272	Benign	4.09	Benign	0.78	Tolerated	3.74	4	-2	-3	4.1	-99.14
c.485G>A	R162H			Uncertain		1	6-33432782-G-A	2	1.24e-6	-9.730	Likely Pathogenic	0.480	Ambiguous	Likely Benign	0.167	Likely Benign										-1.13	Neutral	0.957	Probably Damaging	0.513	Possibly Damaging	4.03	Benign	0.12	Tolerated	3.74	4	2	0	1.3	-19.05
c.48G>A	M16I	Likely Benign		Uncertain		1	6-33420312-G-A	1	6.49e-7	-2.198	Likely Benign	0.722	Likely Pathogenic	Likely Benign	0.057	Likely Benign										-0.15	Neutral	0.000	Benign	0.000	Benign	4.28	Benign	0.00	Affected	4.32	1	2	1	2.6	-18.03
c.491G>A	R164Q			Uncertain		1	6-33432788-G-A	2	1.24e-6	-11.208	Likely Pathogenic	0.600	Likely Pathogenic	Likely Benign	0.184	Likely Benign										-1.86	Neutral	0.957	Probably Damaging	0.342	Benign	3.82	Benign	0.00	Affected	3.74	4	1	1	1.0	-28.06
c.505G>A	D169N			Uncertain		1				-10.713	Likely Pathogenic	0.761	Likely Pathogenic	Likely Benign	0.110	Likely Benign										-2.04	Neutral	0.079	Benign	0.052	Benign	4.07	Benign	0.01	Affected	3.74	4	2	1	0.0	-0.98
c.508C>T	R170W	Likely Pathogenic		Uncertain		2				-11.660	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.241	Likely Benign										-4.28	Deleterious	0.999	Probably Damaging	0.849	Possibly Damaging	3.84	Benign	0.00	Affected	3.74	4	2	-3	3.6	30.03
c.50C>T	S17F	Likely Benign		Uncertain		1	6-33420314-C-T	10	6.49e-6	-3.888	Likely Benign	0.637	Likely Pathogenic	Likely Benign	0.048	Likely Benign										-0.99	Neutral	0.486	Possibly Damaging	0.032	Benign	3.99	Benign	0.00	Affected	4.32	1	-2	-3	3.6	60.10
c.514C>T	R172W	Likely Pathogenic		Uncertain		2	6-33435156-C-T	9	5.58e-6	-10.258	Likely Pathogenic	0.878	Likely Pathogenic	Ambiguous	0.228	Likely Benign										-3.61	Deleterious	0.997	Probably Damaging	0.803	Possibly Damaging	3.95	Benign	0.00	Affected	3.61	5	2	-3	3.6	30.03
c.515G>A	R172Q			Uncertain		1	6-33435157-G-A	3	1.86e-6	-7.245	In-Between	0.465	Ambiguous	Likely Benign	0.135	Likely Benign										-1.72	Neutral	0.804	Possibly Damaging	0.091	Benign	4.04	Benign	0.04	Affected	3.61	5	1	1	1.0	-28.06
c.526A>C	S176R	Likely Benign		Uncertain		1				-6.492	Likely Benign	0.987	Likely Pathogenic	Likely Pathogenic	0.247	Likely Benign										0.94	Neutral	0.718	Possibly Damaging	0.168	Benign	4.16	Benign	0.87	Tolerated			0	-1	-3.7	69.11
c.526A>G	S176G			Uncertain		1	6-33435168-A-G	1	6.20e-7	-7.541	In-Between	0.360	Ambiguous	Likely Benign	0.066	Likely Benign										-1.08	Neutral	0.131	Benign	0.039	Benign	4.08	Benign	0.22	Tolerated	3.54	6	0	1	0.4	-30.03
c.53A>G	Y18C	Likely Benign		Uncertain		1	6-33420317-A-G	44	2.88e-5	-2.658	Likely Benign	0.251	Likely Benign	Likely Benign	0.102	Likely Benign										-0.56	Neutral	0.872	Possibly Damaging	0.206	Benign	4.04	Benign	0.00	Affected	4.32	1	0	-2	3.8	-60.04
c.558G>C	L186F	Likely Pathogenic		Uncertain		1				-11.861	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.132	Likely Benign										-3.03	Deleterious	0.009	Benign	0.012	Benign	3.50	Benign	0.00	Affected			2	0	-1.0	34.02
c.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.59C>G	P20R	Likely Benign		Uncertain		1				-3.548	Likely Benign	0.434	Ambiguous	Likely Benign	0.146	Likely Benign										-0.15	Neutral	0.972	Probably Damaging	0.804	Possibly Damaging	4.33	Benign	0.00	Affected	4.32	1	0	-2	-2.9	59.07
c.59C>T	P20L	Likely Benign		Uncertain		3				-3.289	Likely Benign	0.464	Ambiguous	Likely Benign	0.100	Likely Benign										-0.44	Neutral	0.909	Possibly Damaging	0.713	Possibly Damaging	4.27	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
c.5G>A	S2N	Likely Benign		Uncertain		2	6-33420269-G-A	3	1.96e-6	-4.104	Likely Benign	0.207	Likely Benign	Likely Benign	0.092	Likely Benign										-0.36	Neutral	0.000	Benign	0.000	Benign	4.06	Benign	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.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.662A>G	E221G (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-12.221	Likely Pathogenic	0.992	Likely Pathogenic	Likely Pathogenic	0.863	Likely Pathogenic	1.40	Ambiguous	0.1	1.74	Ambiguous	1.57	Ambiguous	0.71	Ambiguous	-5.56	Deleterious	0.596	Possibly Damaging	0.201	Benign	5.79	Benign	0.00	Affected			0	-2	3.1	-72.06
c.68A>G	D23G	Likely Benign		Uncertain		1				-2.622	Likely Benign	0.684	Likely Pathogenic	Likely Benign	0.100	Likely Benign										-2.45	Neutral	0.805	Possibly Damaging	0.539	Possibly Damaging	3.50	Benign	0.00	Affected			1	-1	3.1	-58.04
c.70G>A	V24I	Likely Benign		Uncertain		1	6-33423479-G-A	9	5.58e-6	-3.701	Likely Benign	0.137	Likely Benign	Likely Benign	0.069	Likely Benign										-0.25	Neutral	0.043	Benign	0.031	Benign	3.96	Benign	0.00	Affected	4.32	1	3	4	0.3	14.03
c.718G>A	D240N	Likely Pathogenic	PH	Uncertain		1				-12.942	Likely Pathogenic	0.755	Likely Pathogenic	Likely Benign	0.701	Likely Pathogenic	0.22	Likely Benign	0.9	0.47	Likely Benign	0.35	Likely Benign	0.37	Likely Benign	-4.37	Deleterious	0.993	Probably Damaging	0.984	Probably Damaging	5.88	Benign	0.01	Affected			2	1	0.0	-0.98
c.719A>G	D240G	Likely Pathogenic	PH	Uncertain		1				-12.825	Likely Pathogenic	0.951	Likely Pathogenic	Ambiguous	0.912	Likely Pathogenic	1.85	Ambiguous	0.1	2.72	Destabilizing	2.29	Destabilizing	0.24	Likely Benign	-6.19	Deleterious	0.993	Probably Damaging	0.984	Probably Damaging	5.79	Benign	0.01	Affected			1	-1	3.1	-58.04
c.73C>T	R25W	Likely Benign		Uncertain		2	6-33423482-C-T	6	3.72e-6	-5.133	Likely Benign	0.549	Ambiguous	Likely Benign	0.158	Likely Benign										-1.60	Neutral	0.994	Probably Damaging	0.919	Probably Damaging	3.92	Benign	0.00	Affected	4.32	1	-3	2	3.6	30.03
c.74G>A	R25Q	Likely Benign		Uncertain		1	6-33423483-G-A	15	9.29e-6	-4.126	Likely Benign	0.212	Likely Benign	Likely Benign	0.038	Likely Benign										-0.70	Neutral	0.829	Possibly Damaging	0.614	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06
c.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.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.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.772C>T	R258C (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437677-C-T	1	6.20e-7	-10.285	Likely Pathogenic	0.790	Likely Pathogenic	Ambiguous	0.771	Likely Pathogenic	1.17	Ambiguous	0.4	1.76	Ambiguous	1.47	Ambiguous	0.87	Ambiguous	-6.79	Deleterious	1.000	Probably Damaging	0.993	Probably Damaging	5.77	Benign	0.00	Affected	3.39	15	-3	-4	7.0	-53.05
c.791T>C	L264P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-12.285	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.767	Likely Pathogenic	5.73	Destabilizing	0.3	6.57	Destabilizing	6.15	Destabilizing	2.65	Destabilizing	-6.43	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.49	Pathogenic	0.00	Affected			-3	-3	-5.4	-16.04
c.82T>C	S28P	Likely Benign		Uncertain		1				-3.309	Likely Benign	0.051	Likely Benign	Likely Benign	0.047	Likely Benign										1.37	Neutral	0.000	Benign	0.000	Benign	4.53	Benign	0.00	Affected	4.32	1	1	-1	-0.8	10.04
c.860A>C	D287A (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-14.686	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.484	Likely Benign	0.30	Likely Benign	0.1	-0.04	Likely Benign	0.13	Likely Benign	0.40	Likely Benign	-7.35	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.58	Pathogenic	0.01	Affected	3.38	23	-2	0	5.3	-44.01
c.700C>T	R234W (3D Viewer)	Likely Pathogenic	PH	Uncertain		1	6-33435551-C-T	3	1.86e-6	-12.625	Likely Pathogenic	0.947	Likely Pathogenic	Ambiguous	0.805	Likely Pathogenic	0.96	Ambiguous	0.3	0.69	Ambiguous	0.83	Ambiguous	0.13	Likely Benign	-5.52	Deleterious	0.997	Probably Damaging	0.803	Possibly Damaging	5.76	Benign	0.01	Affected	3.40	14	2	-3	3.6	30.03	262.8	39.6	-0.1	0.0	-0.2	0.2		X						Potentially Pathogenic	The guanidinium group of Arg234, located in a β-α loop between an anti-parallel β sheet strand (residues Gly227-Phe231) and an α helix (res. Ala236-Val250), forms a salt bridge with the carboxylate group of Glu238 in the α helix. Occasionally, it also bonds with the GAP domain residues Ser678 and Glu680. Thus, the positively charged Arg234 could contribute to the tertiary structure assembly between the PH and GAP domains. In contrast, the indole side chain of Trp234 in the variant is located on the protein surface in the variant simulations and is unable to form any interactions.
c.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.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.866T>C	M289T	Likely Benign	C2	Uncertain		1				-4.668	Likely Benign	0.238	Likely Benign	Likely Benign	0.222	Likely Benign	0.73	Ambiguous	0.1	0.17	Likely Benign	0.45	Likely Benign	-0.01	Likely Benign	-0.47	Neutral	0.801	Possibly Damaging	0.315	Benign	1.83	Pathogenic	0.57	Tolerated			-1	-1	-2.6	-30.09
c.86T>C	M29T	Likely Benign		Uncertain		1				-2.167	Likely Benign	0.122	Likely Benign	Likely Benign	0.199	Likely Benign										-0.37	Neutral	0.018	Benign	0.184	Benign	4.33	Benign	0.00	Affected	4.32	1	-1	-1	-2.6	-30.09
c.878G>A	R293H	Likely Pathogenic	C2	Uncertain		1				-13.009	Likely Pathogenic	0.973	Likely Pathogenic	Likely Pathogenic	0.438	Likely Benign	4.45	Destabilizing	2.3	2.12	Destabilizing	3.29	Destabilizing	0.32	Likely Benign	-4.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.45	Pathogenic	0.04	Affected			2	0	1.3	-19.05
c.88C>T	H30Y	Likely Benign		Uncertain		1				-3.047	Likely Benign	0.115	Likely Benign	Likely Benign	0.082	Likely Benign										-1.84	Neutral	0.273	Benign	0.478	Possibly Damaging	3.99	Benign	0.00	Affected	4.32	1	0	2	1.9	26.03
c.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.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.775C>T	R259W (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-12.186	Likely Pathogenic	0.985	Likely Pathogenic	Likely Pathogenic	0.691	Likely Pathogenic	1.95	Ambiguous	0.8	0.51	Ambiguous	1.23	Ambiguous	0.51	Ambiguous	-7.35	Deleterious	1.000	Probably Damaging	0.993	Probably Damaging	5.76	Benign	0.00	Affected	3.39	15	2	-3	3.6	30.03	254.0	40.0	0.2	0.2	0.2	0.4	X	X					X	Potentially Pathogenic	The guanidinium group of Arg259, located at the beginning of an anti-parallel β sheet strand (res. Arg259-Arg272), forms salt bridges with the carboxylate groups of Asp684 at the end of an α helix (res. Ile683-Gln702, GAP domain) and Asp261 on the same β strand. The Arg259 side chain also frequently forms hydrogen bonds with the backbone carbonyl groups of Ser257, Asn256, and Asp255. In the variant simulations, the indole ring of the Trp259 side chain cannot form salt bridges or maintain hydrogen bonding with the carboxylate group of Asp684 or other nearby residues. Notably, the amino group of the Lys254 side chain maintains a salt bridge with Asp684 and Glu244 throughout the variant simulations, while it forms a cation-π bond with the indole ring of Trp259 in the variant. This salt bridge is not maintained in the WT simulations. Additionally, the partially or loosely α helical conformation of a lysine-containing loop (res. Lys251-Ser257), which extends to a nearby α helix (res. Met414-Asn426), could be stabilized due to the residue swap. Moreover, the bulky size of the Trp259 side chain requires nearby residues to adjust their positioning to accommodate the introduced residue, weakening the tertiary structure assembly between the C2, PH, and GAP domains. The residue swap potentially causes more severe effects during protein folding or for the SynGAP-membrane interaction than the solvent-only simulations imply.
c.791T>A	L264Q (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-15.729	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.678	Likely Pathogenic	3.43	Destabilizing	0.1	2.41	Destabilizing	2.92	Destabilizing	2.48	Destabilizing	-5.52	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	0.49	Pathogenic	0.00	Affected	3.38	18	-2	-2	-7.3	14.97	254.7	-7.6	0.0	0.0	0.0	0.3	X		X				X	Potentially Pathogenic	The iso-butyl branched hydrocarbon side chain of Leu264, located at the end of an anti-parallel β sheet strand (res. Arg259-Arg272), packs against multiple hydrophobic residues such as Leu266, Phe314, Leu317, and Leu323 in the WT simulations. In the variant simulations, the hydrophilic carboxamide group of the Gln264 side chain is not suitable for the hydrophobic niche, causing the hydrophobic residues to make room for the swapped residue. Additionally, the carboxamide group of Gln264 forms hydrogen bonds with the backbone amide groups of Arg405 and Lys256 in the β sheet and the carbonyl group of Val350 in an α helical section of a nearby loop (res. Pro359-Phe358). The residue swap disrupts the packing of the C2 domain, which could adversely affect the C2 domain structure during folding. This disruption could potentially weaken the stability of the SynGAP-membrane association.
c.92G>A	R31Q	Likely Benign		Uncertain		1	6-33423501-G-A	7	4.34e-6	-4.434	Likely Benign	0.136	Likely Benign	Likely Benign	0.051	Likely Benign										-0.92	Neutral	0.829	Possibly Damaging	0.614	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06
c.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.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.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.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.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.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.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.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.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.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.968T>C	L323P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-12.507	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.762	Likely Pathogenic	3.39	Destabilizing	0.6	8.46	Destabilizing	5.93	Destabilizing	2.20	Destabilizing	-4.80	Deleterious	0.999	Probably Damaging	0.977	Probably Damaging	0.59	Pathogenic	0.01	Affected	4.29	398	-3	-3	-5.4	-16.04	201.9	68.2	0.0	0.1	0.6	0.3	X							Potentially Pathogenic	The iso-butyl side chain of Leu323, located at the beginning of an anti-parallel β sheet strand (res. Ala322-Asp330), packs against multiple hydrophobic leucine residues (e.g., Leu264, Leu266, Leu284, Leu286). In contrast, in the variant simulations, the less bulky cyclic five-membered pyrrolidine ring of Pro323 cannot fill the hydrophobic space as effectively as the branched hydrocarbon side chain of leucine. Notably, the backbone amide group of Leu323 forms a hydrogen bond with the backbone carbonyl group of Cys285. Pro323 cannot form this bond due to the absence of the backbone amide group, resulting in partial unfolding of the anti-parallel β sheet end in the variant simulations.
c.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.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.1714T>C	W572R (3D Viewer)	Likely Pathogenic	GAP	Not provided		1				-17.511	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.894	Likely Pathogenic	4.84	Destabilizing	0.1	6.19	Destabilizing	5.52	Destabilizing	1.79	Destabilizing	-12.81	Deleterious					-1.25	Pathogenic	0.00	Affected	3.37	35	2	-3	-3.6	-30.03	312.6	-37.6	0.0	0.0	-1.0	0.0		X	X					Potentially Pathogenic	The indole ring of Trp572, located in an α-helix (res. Arg563-Glu578), lies in a hydrophobic inter-helix space, where it makes extensive hydrophobic interactions with nearby residues such as Met470, Phe569, Leu588, and Ile589. The guanidinium group of Arg572 is similarly sized to the tryptophan it replaced; however, it is also positively charged. In the variant simulations, Arg572 forms hydrogen bonds with other residues in the inter-helix space, such as Ser592 and the backbone carbonyl atom of Leu465. Additionally, Arg572 hydrophobically packs its carbon chain with surrounding residues such as Phe569 and Ile589.However, the introduced residue arginine is too hydrophilic and charged for the hydrophobic space, disrupting the hydrophobic packing of the inter-helix space. Indeed, in the second simulation, Arg572 successfully escapes the hydrophobic niche completely, causing the whole protein to partially unfold.Overall, the residue swap is highly likely to cause critical protein folding problems, as evidenced by the effects seen in the variant simulations.