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.1136C>T	S379L (3D Viewer)	Likely Benign	C2	Benign		1	6-33438041-C-T	8	4.05e-5	-5.641	Likely Benign	0.173	Likely Benign	Likely Benign	0.469	Likely Benign	0.39	Likely Benign	0.2	3.38	Destabilizing	1.89	Ambiguous	-0.52	Ambiguous	-0.85	Neutral	0.015	Benign	0.002	Benign	3.83	Benign	0.04	Affected	4.32	11	-3	-2	4.6	26.08	251.9	-48.1	0.6	1.1	0.0	0.5											Uncertain	Ser379 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like leucine are rarely tolerated. Although no negative structural effects are observed in the variant simulations, Leu379 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1154C>G	S385W (3D Viewer)		C2	Benign		1	6-33438059-C-G			-9.353	Likely Pathogenic	0.362	Ambiguous	Likely Benign	0.373	Likely Benign	0.53	Ambiguous	0.2	0.69	Ambiguous	0.61	Ambiguous	0.00	Likely Benign	-0.84	Neutral	0.986	Probably Damaging	0.968	Probably Damaging	4.63	Benign	0.00	Affected	4.32	3	-2	-3	-0.1	99.14	260.4	-71.2	0.5	1.3	0.7	0.4								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like tryptophan are rarely tolerated. Although no major negative structural effects are observed in the variant simulations, Trp385 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effects on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.	10.1016/j.ajhg.2020.11.011
c.1195G>A	A399T (3D Viewer)	Likely Benign	C2	Benign		1				-5.236	Likely Benign	0.114	Likely Benign	Likely Benign	0.272	Likely Benign	1.24	Ambiguous	0.1	0.91	Ambiguous	1.08	Ambiguous	0.49	Likely Benign	-0.40	Neutral	0.131	Benign	0.039	Benign	5.41	Benign	0.69	Tolerated	3.38	26	1	0	-2.5	30.03	211.4	-41.4	0.0	0.0	0.6	0.4		X						Potentially Pathogenic	The methyl group of Ala399, located in an anti-parallel β sheet strand (res. Ala399-Ile411), is swapped for a hydroxyl-containing threonine. In the variant simulations, the hydroxyl group of Thr399 can form H-bonds with the backbone atoms of the residues in the membrane-facing loops (e.g., Gly382) in the C2 domain. Consequently, the ability of the Thr399 side chain to form H-bonds with the membrane-facing loops could adversely affect the dynamics and stability of the SynGAP-membrane association. However, since the effects on the dynamics of the membrane-facing loops can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1198G>C	V400L (3D Viewer)	Likely Benign	C2	Benign		1	6-33438103-G-C	22	1.36e-5	-1.000	Likely Benign	0.137	Likely Benign	Likely Benign	0.325	Likely Benign	-0.71	Ambiguous	0.2	0.39	Likely Benign	-0.16	Likely Benign	-0.29	Likely Benign	-0.60	Neutral	0.001	Benign	0.001	Benign	5.33	Benign	0.64	Tolerated	3.38	27	2	1	-0.4	14.03	251.0	-30.1	0.0	0.0	0.7	0.1						X		Potentially Benign	The iso-propyl side chain of Val400, located in an anti-parallel β sheet strand (res. Ala399-Ile411), hydrophobically packs against hydrophobic residues within the anti-parallel β sheet of the C2 domain (e.g., Ile268, Ala404, Leu325, Leu402). Val400 is swapped for another hydrophobic residue, leucine, whose branched hydrocarbon side chain is of a comparable size and thus packs favorably within the C2 domain. In short, the residue swap has no apparent negative effect on the structure based on the variant simulations.	10.1016/j.ajhg.2020.11.011
c.1918A>T	T640S (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441177-A-T	1	6.20e-7	-2.371	Likely Benign	0.062	Likely Benign	Likely Benign	0.088	Likely Benign	-0.78	Ambiguous	0.1	0.43	Likely Benign	-0.18	Likely Benign	-0.30	Likely Benign	0.92	Neutral	0.000	Benign	0.001	Benign	3.60	Benign	0.33	Tolerated	3.37	30	1	1	-0.1	-14.03
c.196C>T	P66S	Likely Benign		Benign		1	6-33425804-C-T	2	1.24e-6	-2.760	Likely Benign	0.929	Likely Pathogenic	Ambiguous	0.081	Likely Benign										-1.69	Neutral	0.909	Possibly Damaging	0.641	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
c.1594A>C	T532P (3D Viewer)	Likely Benign	GAP	Benign		1				-2.143	Likely Benign	0.061	Likely Benign	Likely Benign	0.201	Likely Benign	-0.30	Likely Benign	0.2	0.06	Likely Benign	-0.12	Likely Benign	0.08	Likely Benign	-0.90	Neutral	0.005	Benign	0.008	Benign	-1.28	Pathogenic	0.18	Tolerated	3.37	35	0	-1	-0.9	-3.99	174.2	35.1	0.4	0.0	0.1	0.0						X		Potentially Benign	Thr532 is located on an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560) facing the membrane. In the WT simulations, the hydroxyl group of Thr532 occasionally forms hydrogen bonds with the backbone atoms of other loop residues without any specific interaction. In the variant simulations, the Pro532 residue swap does not cause structural changes. Although hydrophilic residues seem more favorable in the loop, the pyrrolidine side chain of proline is well suited for unstructured protein regions such as loops. However, due to its location at the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.
c.1604G>C	S535T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33438847-G-C	14	8.67e-6	-3.886	Likely Benign	0.069	Likely Benign	Likely Benign	0.177	Likely Benign	0.45	Likely Benign	0.1	-0.27	Likely Benign	0.09	Likely Benign	0.17	Likely Benign	-0.81	Neutral	0.000	Benign	0.001	Benign	-1.25	Pathogenic	0.25	Tolerated	3.37	35	1	1	0.1	14.03	201.3	-17.3	-0.1	0.7	-0.2	0.1						X		Potentially Benign	Ser535 is located near the terminal end of an α-helix (res. Ala533-Val560) close to the membrane interface. In the WT simulations, the hydroxyl side chain of Ser535 forms hydrogen bonds with nearby residues (e.g., His539, Glu538) without any specific interactions. These hydrogen bonds disrupt the structure of the terminal end of the α-helix (Ala533-Ser535), causing it to weaken or unfold during the WT simulations. In the variant simulations, Thr535, a hydrophilic residue with a hydroxyl group of almost the same size as Ser, interacts more frequently with the preceding loop residues (e.g., Thr532, Cys531) due to its longer side chain. Regardless, the residue swap is tolerated in the simulations with no negative effects. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.	10.1016/j.ajhg.2020.11.011
c.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.2214T>G	S738R	Likely Benign		Benign		1	6-33441679-T-G	1	6.20e-7	-4.241	Likely Benign	0.570	Likely Pathogenic	Likely Benign	0.068	Likely Benign										-1.55	Neutral	0.473	Possibly Damaging	0.193	Benign	2.69	Benign	0.01	Affected	4.32	2	0	-1	-3.7	69.11
c.1729G>A	A577T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33440781-G-A	6	3.72e-6	-5.311	Likely Benign	0.322	Likely Benign	Likely Benign	0.427	Likely Benign	0.86	Ambiguous	0.1	0.54	Ambiguous	0.70	Ambiguous	0.54	Ambiguous	-1.47	Neutral	0.999	Probably Damaging	0.987	Probably Damaging	-1.31	Pathogenic	0.47	Tolerated	3.37	34	1	0	-2.5	30.03	191.9	-43.4	0.0	0.0	0.7	0.1						X		Potentially Benign	Ala577 is located near the end and outer surface of an α-helix (res. Arg563-Glu578), where its methyl group does not form any particular interactions in the WT simulations. In the variant simulations, the hydroxyl group of the Thr577 side chain hydrogen bonds with the backbone atoms of Arg573 and Lys574 within the same helix, which has the potential to weaken the stability of the secondary structure element. Regardless, the residue swap seems to be well tolerated based on the variant simulations.
c.1742G>A	R581Q (3D Viewer)	Likely Pathogenic	GAP	Benign		1	6-33440794-G-A	8	4.96e-6	-7.584	In-Between	0.673	Likely Pathogenic	Likely Benign	0.481	Likely Benign	1.31	Ambiguous	0.1	-0.42	Likely Benign	0.45	Likely Benign	0.88	Ambiguous	-2.77	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.21	Pathogenic	0.11	Tolerated	3.37	34	1	1	1.0	-28.06	239.6	53.5	-0.2	0.2	-0.4	0.1		X						Potentially Pathogenic	Arg581 is located on a short α-α loop between two α helices (res. Arg563-Glu578 and res. Glu582-Ser604). In the WT simulations, the guanidinium group of Arg581 forms salt bridges with the carboxylate groups of Asp583 within the same helix, as well as with Glu478 and/or Glu480 on a slightly α-helical loop (res. Glu478-Thr488) preceding another α helix (res. Ala461-Phe476).In the variant simulations, the neutral carboxamide group of the Gln581 side chain cannot form any of these salt bridges. Instead, it packs hydrophobically against Met477 and Ile587 or forms hydrogen bonds sporadically with nearby residues (e.g., Asp583, Arg587). Thus, although no drastic changes are observed in the variant simulations, the residue swap could weaken the tertiary structure assembly.
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.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.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.2443C>A	R815S		SH3-binding motif	Benign		1				-7.324	In-Between	0.950	Likely Pathogenic	Ambiguous	0.138	Likely Benign										-1.86	Neutral	0.999	Probably Damaging	0.997	Probably Damaging	2.67	Benign	0.02	Affected			0	-1	3.7	-69.11
c.2014A>G	T672A (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441273-A-G	3	1.86e-6	-6.524	Likely Benign	0.109	Likely Benign	Likely Benign	0.046	Likely Benign	0.51	Ambiguous	0.3	1.15	Ambiguous	0.83	Ambiguous	0.65	Ambiguous	-3.20	Deleterious	0.006	Benign	0.002	Benign	3.44	Benign	0.12	Tolerated	3.40	25	1	0	2.5	-30.03	188.5	42.5	-0.1	0.3	0.2	0.0		X						Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop itself, such as Lys566, Glu666, and Asn669. In the variant simulations, Ala672 can only form a hydrogen bond with Lys566 via its backbone carbonyl group. Consequently, it cannot maintain the Lys566-Glu666 salt bridge through hydrogen bonding, leading to a significant disruption of the intricate and stable hydrogen-bond network between the loop and the helices.
c.2503C>A	L835M	Likely Benign		Benign		1				-4.153	Likely Benign	0.121	Likely Benign	Likely Benign	0.068	Likely Benign										-0.45	Neutral	0.999	Probably Damaging	0.977	Probably Damaging	2.67	Benign	0.12	Tolerated	3.77	5	2	4	-1.9	18.03
c.2578G>A	V860I	Likely Benign		Benign		1	6-33443130-G-A	21	1.30e-5	-4.516	Likely Benign	0.095	Likely Benign	Likely Benign	0.039	Likely Benign										-0.42	Neutral	0.009	Benign	0.006	Benign	4.24	Benign	0.00	Affected	3.77	5	4	3	0.3	14.03
c.2668C>T	R890C			Benign		1	6-33443220-C-T	9	5.58e-6	-5.786	Likely Benign	0.402	Ambiguous	Likely Benign	0.200	Likely Benign										-3.38	Deleterious	1.000	Probably Damaging	0.971	Probably Damaging	3.94	Benign	0.04	Affected	4.32	4	-4	-3	7.0	-53.05
c.2669G>A	R890H	Likely Benign		Benign		1	6-33443221-G-A	19	1.18e-5	-3.600	Likely Benign	0.198	Likely Benign	Likely Benign	0.056	Likely Benign										-1.29	Neutral	0.254	Benign	0.134	Benign	3.97	Benign	0.15	Tolerated	4.32	4	2	0	1.3	-19.05
c.2695A>G	I899V	Likely Benign		Benign		1	6-33443247-A-G	6	3.72e-6	-2.569	Likely Benign	0.074	Likely Benign	Likely Benign	0.040	Likely Benign										0.09	Neutral	0.220	Benign	0.078	Benign	2.75	Benign	0.92	Tolerated	4.32	4	4	3	-0.3	-14.03
c.2743G>A	G915S	Likely Benign		Benign		1	6-33443295-G-A	9	5.58e-6	-3.557	Likely Benign	0.083	Likely Benign	Likely Benign	0.050	Likely Benign										-0.88	Neutral	0.801	Possibly Damaging	0.201	Benign	2.73	Benign	0.31	Tolerated	3.77	5	1	0	-0.4	30.03
c.2765G>A	R922Q	Likely Benign		Benign		1	6-33443317-G-A	7	4.34e-6	-3.295	Likely Benign	0.189	Likely Benign	Likely Benign	0.085	Likely Benign										-0.27	Neutral	0.992	Probably Damaging	0.736	Possibly Damaging	2.57	Benign	0.20	Tolerated	3.77	5	1	1	1.0	-28.06
c.280C>T	P94S	Likely Benign		Benign		1	6-33425888-C-T	5	3.10e-6	-3.151	Likely Benign	0.084	Likely Benign	Likely Benign	0.093	Likely Benign										-2.36	Neutral	0.092	Benign	0.008	Benign	4.13	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
c.2818G>C	G940R	Likely Benign		Benign		1	6-33443370-G-C	5	3.10e-6	-6.169	Likely Benign	0.480	Ambiguous	Likely Benign	0.060	Likely Benign										0.02	Neutral	0.922	Possibly Damaging	0.543	Possibly Damaging	2.73	Benign	0.15	Tolerated	3.77	5	-3	-2	-4.1	99.14
c.2830G>A	G944S	Likely Benign		Benign		1	6-33443382-G-A	13	8.05e-6	-5.303	Likely Benign	0.082	Likely Benign	Likely Benign	0.223	Likely Benign										-0.75	Neutral	0.007	Benign	0.004	Benign	3.77	Benign	0.00	Affected	4.32	4	1	0	-0.4	30.03
c.2837G>A	G946E	Likely Benign		Benign		3	6-33443389-G-A	13	8.05e-6	-8.793	Likely Pathogenic	0.257	Likely Benign	Likely Benign	0.341	Likely Benign										-0.51	Neutral	0.818	Possibly Damaging	0.355	Benign	4.58	Benign	0.00	Affected	4.32	4	0	-2	-3.1	72.06
c.2873A>C	H958P	Likely Benign		Benign		1	6-33443425-A-C	2	1.24e-6	-8.369	Likely Pathogenic	0.068	Likely Benign	Likely Benign	0.204	Likely Benign										-0.36	Neutral	0.925	Possibly Damaging	0.316	Benign	4.14	Benign	0.10	Tolerated	3.77	5	0	-2	1.6	-40.02
c.2909A>G	E970G	Likely Benign		Benign		1				-0.167	Likely Benign	0.139	Likely Benign	Likely Benign	0.139	Likely Benign										-0.93	Neutral	0.144	Benign	0.058	Benign	4.09	Benign	0.10	Tolerated	4.32	2	0	-2	3.1	-72.06
c.2987C>G	P996R	Likely Benign		Benign		1				-4.457	Likely Benign	0.141	Likely Benign	Likely Benign	0.040	Likely Benign										-1.04	Neutral	0.144	Benign	0.085	Benign	4.26	Benign	0.01	Affected	4.32	4	-2	0	-2.9	59.07
c.3020G>A	S1007N	Likely Benign		Benign		1				-5.113	Likely Benign	0.803	Likely Pathogenic	Ambiguous	0.075	Likely Benign										-1.54	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.65	Benign	0.01	Affected	3.77	5	1	1	-2.7	27.03
c.3049T>C	F1017L	Likely Benign		Benign		1				-2.048	Likely Benign	0.934	Likely Pathogenic	Ambiguous	0.157	Likely Benign										-2.38	Neutral	0.798	Possibly Damaging	0.373	Benign	2.65	Benign	0.72	Tolerated	3.77	5	0	2	1.0	-34.02
c.3100C>G	P1034A	Likely Benign		Benign		1				-4.174	Likely Benign	0.178	Likely Benign	Likely Benign	0.060	Likely Benign										-2.44	Neutral	0.059	Benign	0.061	Benign	2.47	Pathogenic	0.06	Tolerated	3.77	5	1	-1	3.4	-26.04
c.311G>T	R104L	Likely Benign		Benign		1	6-33432176-G-T	1	6.20e-7	-3.563	Likely Benign	0.578	Likely Pathogenic	Likely Benign	0.170	Likely Benign										-1.38	Neutral	0.001	Benign	0.002	Benign	4.05	Benign	0.00	Affected	4.32	1	-2	-3	8.3	-43.03
c.3152G>A	G1051D			Benign		1	6-33443704-G-A	2	1.24e-6	-9.379	Likely Pathogenic	0.311	Likely Benign	Likely Benign	0.445	Likely Benign										-0.31	Neutral	0.761	Possibly Damaging	0.239	Benign	-0.74	Pathogenic	0.39	Tolerated	3.77	5	-1	1	-3.1	58.04
c.3160G>A	G1054S	Likely Benign		Benign		1	6-33443712-G-A	32	1.99e-5	-5.294	Likely Benign	0.075	Likely Benign	Likely Benign	0.160	Likely Benign										0.21	Neutral	0.121	Benign	0.013	Benign	4.04	Benign	0.63	Tolerated	3.77	5	1	0	-0.4	30.03
c.3179G>T	G1060V	Likely Benign		Benign		1	6-33443731-G-T	1	6.22e-7	-6.966	Likely Benign	0.103	Likely Benign	Likely Benign	0.369	Likely Benign										-0.73	Neutral	0.986	Probably Damaging	0.728	Possibly Damaging	2.63	Benign	0.33	Tolerated	4.32	2	-1	-3	4.6	42.08
c.3237C>G	S1079R	Likely Benign		Benign		1				-4.579	Likely Benign	0.955	Likely Pathogenic	Ambiguous	0.124	Likely Benign										-1.81	Neutral	0.177	Benign	0.075	Benign	3.86	Benign	0.00	Affected	3.77	5	0	-1	-3.7	69.11
c.3290C>T	P1097L	Likely Benign		Benign		1				-4.410	Likely Benign	0.145	Likely Benign	Likely Benign	0.131	Likely Benign										-2.07	Neutral	0.611	Possibly Damaging	0.198	Benign	2.64	Benign	0.05	Affected	3.77	5	-3	-3	5.4	16.04
c.3305C>T	A1102V	Likely Benign		Benign		1	6-33443857-C-T			-2.440	Likely Benign	0.077	Likely Benign	Likely Benign	0.081	Likely Benign										-1.27	Neutral	0.017	Benign	0.028	Benign	2.29	Pathogenic	0.12	Tolerated	3.77	5	0	0	2.4	28.05
c.3310C>T	P1104S	Likely Benign		Benign		1	6-33443862-C-T	1	6.54e-7	-2.330	Likely Benign	0.073	Likely Benign	Likely Benign	0.088	Likely Benign										-0.30	Neutral	0.770	Possibly Damaging	0.404	Benign	2.77	Benign	0.10	Tolerated	3.77	5	-1	1	0.8	-10.04
c.3344T>C	I1115T	Likely Benign		Benign		9	6-33443896-T-C	20536	1.36e-2	-2.670	Likely Benign	0.068	Likely Benign	Likely Benign	0.100	Likely Benign										-0.04	Neutral	0.000	Benign	0.001	Benign	2.76	Benign	0.23	Tolerated	4.32	2	0	-1	-5.2	-12.05
c.3355G>A	G1119R			Benign		1	6-33443907-G-A	64	4.23e-5	-8.489	Likely Pathogenic	0.473	Ambiguous	Likely Benign	0.303	Likely Benign										0.10	Neutral	0.969	Probably Damaging	0.462	Possibly Damaging	4.03	Benign	0.10	Tolerated	4.32	2	-3	-2	-4.1	99.14
c.3397A>G	I1133V	Likely Benign		Benign		1	6-33443949-A-G	22	1.48e-5	-3.362	Likely Benign	0.067	Likely Benign	Likely Benign	0.180	Likely Benign										0.06	Neutral	0.007	Benign	0.007	Benign	5.47	Benign	0.58	Tolerated	4.32	3	4	3	-0.3	-14.03																10.1016/j.ajhg.2020.11.011
c.3410A>C	H1137P	Likely Benign		Benign		1	6-33444445-A-C	12	7.44e-6	-2.098	Likely Benign	0.054	Likely Benign	Likely Benign	0.419	Likely Benign										-1.93	Neutral	0.925	Possibly Damaging	0.703	Possibly Damaging	5.29	Benign	0.00	Affected	4.32	4	-2	0	1.6	-40.02
c.3631A>G	M1211V	Likely Benign	Coiled-coil	Benign		1	6-33446623-A-G	3	1.86e-6	-2.101	Likely Benign	0.258	Likely Benign	Likely Benign	0.412	Likely Benign										-0.29	Neutral	0.932	Possibly Damaging	0.949	Probably Damaging	5.43	Benign	0.72	Tolerated	3.77	5	1	2	2.3	-32.06
c.3638A>G	N1213S	Likely Benign	Coiled-coil	Benign		1	6-33446630-A-G	13	8.05e-6	-4.086	Likely Benign	0.081	Likely Benign	Likely Benign	0.094	Likely Benign										-0.56	Neutral	0.906	Possibly Damaging	0.551	Possibly Damaging	2.82	Benign	0.68	Tolerated	3.77	5	1	1	2.7	-27.03																10.1016/j.ajhg.2020.11.011
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.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.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.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.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.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.407G>A	R136Q			Benign		1	6-33432704-G-A	13	9.17e-6	-11.146	Likely Pathogenic	0.950	Likely Pathogenic	Ambiguous	0.190	Likely Benign										-2.26	Neutral	0.957	Probably Damaging	0.342	Benign	3.52	Benign	0.01	Affected	3.61	5	1	1	1.0	-28.06
c.502C>T	H168Y	Likely Benign		Benign		1				-8.914	Likely Pathogenic	0.264	Likely Benign	Likely Benign	0.065	Likely Benign										-1.53	Neutral	0.192	Benign	0.062	Benign	4.18	Benign	0.01	Affected	4.32	3	0	2	1.9	26.03
c.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.76G>A	G26R	Likely Benign		Benign		1	6-33423485-G-A	3	1.86e-6	-2.946	Likely Benign	0.678	Likely Pathogenic	Likely Benign	0.189	Likely Benign										-2.22	Neutral	0.994	Probably Damaging	0.990	Probably Damaging	3.87	Benign	0.00	Affected	4.32	1	-3	-2	-4.1	99.14
c.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.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.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.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.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.1730C>G	A577G (3D Viewer)	Likely Benign	GAP	Benign/Likely benign		2	6-33440782-C-G	1	6.20e-7	-5.717	Likely Benign	0.268	Likely Benign	Likely Benign	0.443	Likely Benign	0.83	Ambiguous	0.0	1.02	Ambiguous	0.93	Ambiguous	0.86	Ambiguous	-1.84	Neutral	0.997	Probably Damaging	0.990	Probably Damaging	-1.31	Pathogenic	0.31	Tolerated	3.37	34	1	0	-2.2	-14.03	158.7	23.6	0.0	0.0	0.0	0.0						X		Potentially Benign	Ala577 is located near the end and outer surface of an α-helix (res. Arg563-Glu578), where its methyl group does not form any particular interactions in the WT simulations. The introduced residue, glycine, is known as an “α-helix breaker.” However, the residue swap caused only minor helix shortening in one of the replica simulations for the variant system. Regardless, the residue swap seems to be well tolerated based on the variant simulations.
c.223G>A	E75K	Likely Benign		Benign/Likely benign		2				-4.020	Likely Benign	0.358	Ambiguous	Likely Benign	0.134	Likely Benign										-1.12	Neutral	0.748	Possibly Damaging	0.017	Benign	4.07	Benign	0.00	Affected			0	1	-0.4	-0.94
c.2381C>T	P794L	Likely Benign	SH3-binding motif	Benign/Likely benign		2	6-33442933-C-T	73	4.52e-5	-3.808	Likely Benign	0.079	Likely Benign	Likely Benign	0.075	Likely Benign										-0.80	Neutral	0.761	Possibly Damaging	0.321	Benign	4.24	Benign	0.03	Affected	4.07	3	-3	-3	5.4	16.04
c.1888A>G	I630V (3D Viewer)		GAP	Benign/Likely benign		4	6-33440940-A-G	59	3.66e-5	-7.264	In-Between	0.145	Likely Benign	Likely Benign	0.143	Likely Benign	1.33	Ambiguous	0.0	0.94	Ambiguous	1.14	Ambiguous	0.64	Ambiguous	-0.38	Neutral	0.018	Benign	0.011	Benign	-1.37	Pathogenic	0.35	Tolerated	3.37	34	4	3	-0.3	-14.03	235.0	26.2	-0.1	0.0	-0.3	0.1						X		Potentially Benign	The sec-butyl side chain of Ile630, located in an α helix (res. Glu617-Asn635), packs with hydrophobic residues (e.g., Phe594, Leu633, Ile626, Ile602) in the hydrophobic inter-helix space between two α helices (res. Glu617-Asn635 and res. Glu582-Met603).In the variant simulations, the iso-propyl side chain of Val630, which shares a similar size and physicochemical properties with Ile630 in the WT, maintains similar interactions in the inter-helix space. Although no negative structural effects are observed during the simulations, the implications of the residue swap on the complex formation with the GTPase, due to its location, cannot be investigated using solvent-only simulations.
c.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.2845G>A	G949S	Likely Benign		Benign/Likely benign		4	6-33443397-G-A	122	7.56e-5	-5.693	Likely Benign	0.072	Likely Benign	Likely Benign	0.321	Likely Benign										0.30	Neutral	0.611	Possibly Damaging	0.102	Benign	2.23	Pathogenic	0.00	Affected	4.32	4	1	0	-0.4	30.03																10.1016/j.ajhg.2020.11.011
c.2900G>A	R967Q	Likely Benign		Benign/Likely benign		2	6-33443452-G-A	31	1.92e-5	-3.057	Likely Benign	0.080	Likely Benign	Likely Benign	0.104	Likely Benign										-0.01	Neutral	0.994	Probably Damaging	0.626	Possibly Damaging	4.21	Benign	0.36	Tolerated	4.32	2	1	1	1.0	-28.06
c.3134C>G	A1045G	Likely Benign		Benign/Likely benign		7	6-33443686-C-G	1407	8.72e-4	-3.246	Likely Benign	0.075	Likely Benign	Likely Benign	0.024	Likely Benign										-1.21	Neutral	0.224	Benign	0.066	Benign	2.64	Benign	0.33	Tolerated	3.77	5	1	0	-2.2	-14.03																10.1016/j.ajhg.2020.11.011
c.3308G>A	R1103H	Likely Benign		Benign/Likely benign		3	6-33443860-G-A	31	2.03e-5	-3.622	Likely Benign	0.156	Likely Benign	Likely Benign	0.116	Likely Benign										-1.97	Neutral	0.996	Probably Damaging	0.733	Possibly Damaging	2.49	Pathogenic	0.01	Affected	3.77	5	2	0	1.3	-19.05
c.3364G>A	G1122S	Likely Benign		Benign/Likely benign		2	6-33443916-G-A	27	1.79e-5	-4.880	Likely Benign	0.072	Likely Benign	Likely Benign	0.189	Likely Benign										-0.08	Neutral	0.022	Benign	0.006	Benign	4.89	Benign	0.92	Tolerated	3.77	5	1	0	-0.4	30.03
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.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.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.1055C>A	T352N (3D Viewer)	Likely Benign	C2	Likely Benign		1	6-33437960-C-A	2	1.24e-6	-4.817	Likely Benign	0.117	Likely Benign	Likely Benign	0.027	Likely Benign	0.20	Likely Benign	0.0	-0.04	Likely Benign	0.08	Likely Benign	0.45	Likely Benign	-0.92	Neutral	0.255	Benign	0.057	Benign	1.75	Pathogenic	0.19	Tolerated	3.37	25	0	0	-2.8	13.00	208.4	-14.5	-0.2	0.1	-0.1	0.0		X						Potentially Benign	Thr352 is located in a short α helical section within a loop connecting two β strands (res. Gly341-Pro349, res. Thr359-Pro364) originating from two different anti-parallel β sheets of the C2 domain. In the WT simulations, the side chain hydroxyl and backbone amide groups of Thr354 form hydrogen bonds with the backbone carbonyl group of Pro349 at the end of the preceding β strand. This arrangement likely stabilizes the α helical section and aids in folding, keeping the short secondary structure element intact in the variant simulations. However, the carboxamide group of the Asn352 side chain does not form hydrogen bonds with the backbone carbonyl group of Pro349. Instead, it packs against the cyclic ring and forms hydrogen bonds with the phenol group of the Tyr363 side chain in the other β strand.
c.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.1231A>G	I411V (3D Viewer)	Likely Benign	GAP	Likely Benign		1				-6.290	Likely Benign	0.385	Ambiguous	Likely Benign	0.212	Likely Benign	0.74	Ambiguous	0.0	0.82	Ambiguous	0.78	Ambiguous	0.99	Ambiguous	-0.86	Neutral	0.935	Possibly Damaging	0.858	Possibly Damaging	3.90	Benign	0.27	Tolerated	3.38	28	4	3	-0.3	-14.03	233.3	28.2	-0.2	0.0	-0.2	0.0						X		Potentially Benign	The sec-butyl side chain of Ile411, located in the hydrophobic space between an anti-parallel β sheet strand (res. Pro398-Ile411) and an α helix (res. Asp684-Gln702), packs against multiple residues (e.g., Met409, Arg259). In the variant simulations, the side chain of Val411 is able to favorably fill the same hydrophobic niche despite its slightly smaller size. In short, the residue swap has no apparent negative effect on the structure based on the simulations.
c.140G>A	R47Q	Likely Benign		Likely Benign		1	6-33423549-G-A	4	2.48e-6	-4.989	Likely Benign	0.347	Ambiguous	Likely Benign	0.096	Likely Benign										-0.57	Neutral	0.829	Possibly Damaging	0.614	Possibly Damaging	4.12	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.1172G>T	G391V (3D Viewer)	Likely Benign	C2	Likely Benign		1	6-33438077-G-T	3	1.86e-6	-6.642	Likely Benign	0.133	Likely Benign	Likely Benign	0.595	Likely Pathogenic	4.23	Destabilizing	1.3	4.81	Destabilizing	4.52	Destabilizing	-0.11	Likely Benign	-0.98	Neutral	0.994	Probably Damaging	0.887	Possibly Damaging	1.32	Pathogenic	0.10	Tolerated	3.69	8	-1	-3	4.6	42.08	228.6	-69.0	0.0	0.8	-0.5	0.3								Uncertain	Gly387 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364 and res. Ala399-Ile411). The Ω loop is assumed to directly interact with the membrane, and it is observed to move arbitrarily throughout the WT solvent simulations. This loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play significant roles in protein functions that require flexibility, and thus hydrophobic residues like valine are rarely tolerated. Although no negative structural effects are visualized in the variant’s simulations, Val391 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. Since the effects on the Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1454G>A	R485H (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438486-G-A	13	8.05e-6	-13.628	Likely Pathogenic	0.948	Likely Pathogenic	Ambiguous	0.618	Likely Pathogenic	0.77	Ambiguous	0.1	0.12	Likely Benign	0.45	Likely Benign	1.13	Destabilizing	-4.97	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.93	Pathogenic	0.00	Affected	3.37	35	0	2	1.3	-19.05
c.1610C>T	A537V (3D Viewer)	Likely Benign	GAP	Likely Benign		1	6-33438853-C-T	7	4.34e-6	-6.888	Likely Benign	0.120	Likely Benign	Likely Benign	0.382	Likely Benign	0.54	Ambiguous	0.0	-0.05	Likely Benign	0.25	Likely Benign	0.41	Likely Benign	-1.97	Neutral	0.977	Probably Damaging	0.469	Possibly Damaging	-1.26	Pathogenic	0.24	Tolerated	3.37	35	0	0	2.4	28.05	220.3	-45.1	0.0	0.0	-0.7	0.1						X		Potentially Benign	Ala537 is located on the outer surface of an α-helix (res. Ala533-Val560). The methyl group of Ala537 is on the surface and does not form any interactions. In the variant simulations, the iso-propyl side chain of Val537 is also on the surface, similar to Ala537 in the WT, causing no negative structural effects.
c.1667A>T	N556I (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438910-A-T			-13.391	Likely Pathogenic	0.929	Likely Pathogenic	Ambiguous	0.761	Likely Pathogenic	0.64	Ambiguous	0.0	0.17	Likely Benign	0.41	Likely Benign	0.26	Likely Benign	-7.52	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.35	Pathogenic	0.02	Affected	3.37	35	-3	-2	8.0	-0.94
c.1408A>C	M470L (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438440-A-C	1	6.20e-7	-8.993	Likely Pathogenic	0.406	Ambiguous	Likely Benign	0.678	Likely Pathogenic	0.73	Ambiguous	0.1	0.84	Ambiguous	0.79	Ambiguous	1.04	Destabilizing	-2.72	Deleterious	0.484	Possibly Damaging	0.654	Possibly Damaging	-1.22	Pathogenic	0.16	Tolerated	3.37	34	4	2	1.9	-18.03	225.3	17.9	0.0	0.0	-0.8	0.5						X		Potentially Benign	The thioether group of Met470, located in the middle of an α helix (res. Ala461–Phe476), interacts with hydrophobic residues in the inter-helix space (e.g., Val473, Leu558) formed by two other α helices (res. Ser604–Arg581, res. Pro562–Arg579). In the WT simulations, Met470 also packs against the positively charged guanidinium groups of Arg575, Arg429, and Arg579, which form salt bridges with the negatively charged carboxylate groups of the Asp474 and Asp467 side chains at the protein surface. In the variant simulations, the iso-butyl side chain of Leu470 packs similarly with the hydrophobic residues as methionine, resulting in no negative effects on the protein structure during the simulation.
c.1441C>T	H481Y (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33438473-C-T	16	9.91e-6	-10.910	Likely Pathogenic	0.565	Likely Pathogenic	Likely Benign	0.256	Likely Benign	-0.53	Ambiguous	0.1	-0.46	Likely Benign	-0.50	Ambiguous	0.20	Likely Benign	-3.32	Deleterious	0.988	Probably Damaging	0.979	Probably Damaging	3.40	Benign	0.59	Tolerated	3.37	33	0	2	1.9	26.03	256.5	-44.4	0.0	0.0	0.2	0.2	X			X				Uncertain	The imidazole ring of the His481 side chain is located in a short helical structure (res. Glu480-Leu482) within an α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. In the WT simulations, His481 alternately stacks against Arg485, Arg587, and Glu480 without a definite role. In the variant simulations, Tyr481 also alternately stacks with nearby arginine residues, including Arg485, Arg587, and Arg479. The interaction between Tyr481 and Arg479 affects the α-α loop, causing it to fold into a distorted helical structure, an effect that might be more pronounced during protein folding. Finally, the potential effect of the residue swap on SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.1625A>G	N542S (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1				-9.675	Likely Pathogenic	0.767	Likely Pathogenic	Likely Benign	0.752	Likely Pathogenic	0.98	Ambiguous	0.1	0.99	Ambiguous	0.99	Ambiguous	0.91	Ambiguous	-4.40	Deleterious	1.000	Probably Damaging	0.989	Probably Damaging	-1.36	Pathogenic	0.13	Tolerated	3.37	35	1	1	2.7	-27.03	212.5	32.1	0.0	0.0	-0.6	0.3		X						Potentially Pathogenic	Asn542 is located in an α-helix (res. Ala533-Val560) next to an α-α loop between two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the carboxamide group of the Asn542 side chain forms a hydrogen bond with the backbone carbonyl group of Asn523 and packs favourably against Glu522 from the loop. In contrast, in the variant simulations, the hydroxyl group of the Ser542 side chain is unable to maintain either the hydrogen bond with Asn523 or the packing against the Glu522 side chain. Instead, the hydroxyl group of Ser542 occasionally forms a hydrogen bond with the backbone carbonyl group of Glu538.Altogether, the residue swap results in a looser helix-loop association, which is especially evident in the third replica simulation, where Asn523 moves away from its initial placement next to the α-helix. In short, based on the simulations, the residue swap weakens the GAP domain tertiary structure assembly, which in turn could negatively affect protein folding.
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.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.2245C>T	R749W			Likely Benign		1	6-33441710-C-T	3	1.86e-6	-7.647	In-Between	0.338	Likely Benign	Likely Benign	0.173	Likely Benign										-2.62	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.59	Benign	0.00	Affected	4.32	2	2	-3	3.6	30.03
c.2246G>A	R749Q	Likely Benign		Likely Benign		1	6-33441711-G-A	4	2.48e-6	-3.069	Likely Benign	0.212	Likely Benign	Likely Benign	0.152	Likely Benign										-1.00	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	2.64	Benign	0.03	Affected	4.32	2	1	1	1.0	-28.06
c.1787G>A	R596H (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33440839-G-A	15	9.29e-6	-11.128	Likely Pathogenic	0.950	Likely Pathogenic	Ambiguous	0.717	Likely Pathogenic	3.00	Destabilizing	0.9	0.43	Likely Benign	1.72	Ambiguous	1.35	Destabilizing	-4.97	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.43	Pathogenic	0.00	Affected	3.37	35	2	0	1.3	-19.05	223.5	80.5	-0.1	0.0	-0.1	0.3		X		X				Potentially Pathogenic	The guanidinium group of Arg596, located in an α helix (res. Glu582-Met603), forms a salt bridge with the carboxylate group of Glu495 from another α helix (res. Leu489-Glu519). In the WT simulations, the side chain of Arg596 hydrogen bonds with the backbone carbonyl groups of Asn487, Glu486, Arg485, and Phe484. Additionally, Arg596 can hydrogen bond with the carboxamide group of the Asn487 side chain on an opposing loop that links two α helices (res. Ala461-Arg475, res. Leu489-Glu519).In the variant simulations, the imidazole ring of His596 can form hydrogen bonds with the same residues as arginine; however, these interactions are not as coordinated or strong in comparison. Thus, the residue swap could affect the tertiary structure assembly more profoundly than observed in the simulations. Notably, Arg596 plays a key role in positioning the aforementioned loop, which is crucial for the placement of the “arginine finger” or the Arg485 side chain during RasGTPase activation.
c.2359C>A	P787T	Likely Pathogenic	SH3-binding motif	Likely Benign		1	6-33442911-C-A	17	1.05e-5	-4.813	Likely Benign	0.603	Likely Pathogenic	Likely Benign	0.258	Likely Benign										-4.40	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.46	Pathogenic	0.01	Affected	3.64	6	0	-1	0.9	3.99
c.1862G>A	R621Q (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33440914-G-A	19	1.18e-5	-14.682	Likely Pathogenic	0.910	Likely Pathogenic	Ambiguous	0.621	Likely Pathogenic	0.81	Ambiguous	0.1	1.13	Ambiguous	0.97	Ambiguous	1.35	Destabilizing	-3.98	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	2.82	Benign	0.01	Affected	3.37	35	1	1	1.0	-28.06	243.7	54.3	0.0	0.0	-0.4	0.2		X		X				Potentially Pathogenic	The guanidinium group of Arg621, located in an α helix (res. Glu617-Asn635), forms a salt bridge with Glu525 in a nearby loop and stacks with Leu635. In the variant simulations, the carboxamide side chain of Gln621, which can act as both a hydrogen bond acceptor and donor, also stacks with Leu635 but can only sporadically hydrogen bond with Glu525.Accordingly, the residue swap could affect the tertiary structure integrity by disrupting the salt bridge formation. Additionally, due to its location at the GAP-Ras interface, the residue swap could impact the complex formation with the GTPase, but this cannot be investigated using solvent-only simulations.
c.1991T>C	L664S (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441250-T-C	1	6.20e-7	-16.498	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.543	Likely Pathogenic	3.75	Destabilizing	0.2	3.63	Destabilizing	3.69	Destabilizing	2.77	Destabilizing	-5.99	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	2.85	Benign	0.00	Affected	3.38	28	-3	-2	-4.6	-26.08	215.5	50.1	0.0	0.0	-0.2	0.2						X		Potentially Benign	The iso-butyl side chain of L664, located on an α-helix (res. Ser641-Glu666), hydrophobically interacts with residues in the inter-helix space between three helices (res. Glu617-Asn635, res. Glu582-Met603, and res. Ser641-Glu666), such as Ile589, Phe663, and Met660. In the variant simulations, the hydroxyl group of Ser664 forms hydrogen bonds with the backbone carbonyl oxygen of another helix residue, such as Met660 or Gln661. This interaction is known to destabilize hydrogen bonding in the α-helix, but this effect was not observed in the simulations. Additionally, Ser664 occasionally forms hydrogen bonds with the carboxylate group of Asp586 on another α-helix (res. Glu582-Met603), which could minimally influence the tertiary structure assembly. Despite these interactions, no major negative effects on the protein structure were observed during the simulations.
c.1997A>G	E666G (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441256-A-G	10	6.20e-6	-12.261	Likely Pathogenic	0.911	Likely Pathogenic	Ambiguous	0.522	Likely Pathogenic	1.57	Ambiguous	0.1	1.46	Ambiguous	1.52	Ambiguous	0.93	Ambiguous	-6.25	Deleterious	1.000	Probably Damaging	0.970	Probably Damaging	3.37	Benign	0.02	Affected	3.38	28	0	-2	3.1	-72.06	173.9	98.5	0.0	0.0	-0.7	0.0		X						Potentially Pathogenic	In the WT simulations, the carboxylate group of Glu666, located on the α-helix (res. Ser641-Glu666), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), such as Lys566, Thr672, and Asn669. In the variant simulations, the carbonyl group of Gly666 occasionally forms hydrogen bonds with Lys566 and Asn669. However, Gly666 lacks a side chain and thus cannot maintain as well-coordinated a hydrogen-bond network as Glu666 in the WT, which may affect the tertiary structure assembly.
c.2444G>A	R815H		SH3-binding motif	Likely Benign		2	6-33442996-G-A	24	1.49e-5	-7.474	In-Between	0.553	Ambiguous	Likely Benign	0.157	Likely Benign										-1.81	Neutral	1.000	Probably Damaging	0.998	Probably Damaging	2.61	Benign	0.02	Affected	4.32	4	2	0	1.3	-19.05																10.1016/j.ajhg.2020.11.011
c.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.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.2635_2636delinsAA	A879K	Likely Benign		Likely Benign		1				-5.877	Likely Benign	0.757	Likely Pathogenic	Likely Benign												-0.71	Neutral	0.969	Probably Damaging	0.593	Possibly Damaging	2.69	Benign	0.21	Tolerated	3.77	5	-1	-1	-5.7	57.10
c.2669G>C	R890P	Likely Benign		Likely Benign		2	6-33443221-G-C	28	1.74e-5	-1.931	Likely Benign	0.301	Likely Benign	Likely Benign	0.191	Likely Benign										-1.21	Neutral	0.999	Probably Damaging	0.977	Probably Damaging	4.02	Benign	0.28	Tolerated	4.32	4	0	-2	2.9	-59.07
c.2704G>A	A902T	Likely Benign		Likely Benign		1	6-33443256-G-A	36	2.23e-5	-4.966	Likely Benign	0.116	Likely Benign	Likely Benign	0.075	Likely Benign										-1.11	Neutral	0.951	Possibly Damaging	0.617	Possibly Damaging	2.61	Benign	0.01	Affected	3.77	5	1	0	-2.5	30.03
c.2710A>G	M904V	Likely Benign		Likely Benign		2	6-33443262-A-G	77	4.78e-5	-2.907	Likely Benign	0.112	Likely Benign	Likely Benign	0.058	Likely Benign										-0.33	Neutral	0.039	Benign	0.023	Benign	2.80	Benign	0.10	Tolerated	3.77	5	2	1	2.3	-32.06
c.2719A>T	S907C	Likely Benign		Likely Benign		1				-6.685	Likely Benign	0.298	Likely Benign	Likely Benign	0.113	Likely Benign										-2.34	Neutral	0.999	Probably Damaging	0.988	Probably Damaging	2.60	Benign	0.02	Affected	3.77	5	0	-1	3.3	16.06
c.272A>G	E91G	Likely Benign		Likely Benign		1				-3.226	Likely Benign	0.783	Likely Pathogenic	Likely Benign	0.110	Likely Benign										-2.18	Neutral	0.947	Possibly Damaging	0.727	Possibly Damaging	3.86	Benign	0.00	Affected	4.32	1	0	-2	3.1	-72.06
c.2840G>C	G947A	Likely Benign		Likely Benign		1	6-33443392-G-C	28	1.73e-5	-6.511	Likely Benign	0.080	Likely Benign	Likely Benign	0.156	Likely Benign										-0.41	Neutral	0.224	Benign	0.131	Benign	4.97	Benign	0.10	Tolerated	4.32	4	1	0	2.2	14.03
c.2860C>T	P954S	Likely Benign		Likely Benign		1	6-33443412-C-T	1	6.20e-7	-3.525	Likely Benign	0.062	Likely Benign	Likely Benign	0.143	Likely Benign										-0.25	Neutral	0.954	Possibly Damaging	0.812	Possibly Damaging	2.87	Benign	1.00	Tolerated	3.77	5	1	-1	0.8	-10.04
c.2945A>G	Y982C	Likely Benign		Likely Benign		1	6-33443497-A-G	2	1.24e-6	-6.256	Likely Benign	0.746	Likely Pathogenic	Likely Benign	0.195	Likely Benign										-1.67	Neutral	0.997	Probably Damaging	0.923	Probably Damaging	3.87	Benign	0.00	Affected	4.32	1	0	-2	3.8	-60.04
c.2948G>A	S983N			Likely Benign		1	6-33443500-G-A	6	3.72e-6	-5.604	Likely Benign	0.909	Likely Pathogenic	Ambiguous	0.136	Likely Benign										-1.78	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.04	Pathogenic	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.3022G>A	D1008N	Likely Benign		Likely Benign		1	6-33443574-G-A	3	1.86e-6	-4.045	Likely Benign	0.714	Likely Pathogenic	Likely Benign	0.128	Likely Benign										-2.15	Neutral	0.999	Probably Damaging	0.997	Probably Damaging	2.75	Benign	0.01	Affected	3.77	5	2	1	0.0	-0.98
c.3048C>A	D1016E	Likely Benign		Likely Benign		1	6-33443600-C-A	2	1.24e-6	-3.422	Likely Benign	0.216	Likely Benign	Likely Benign	0.017	Likely Benign										-0.37	Neutral	0.008	Benign	0.028	Benign	2.64	Benign	0.65	Tolerated	3.77	5	2	3	0.0	14.03
c.3194C>T	P1065L			Likely Benign		1	6-33443746-C-T	14	8.71e-6	-5.085	Likely Benign	0.089	Likely Benign	Likely Benign	0.068	Likely Benign										-2.94	Deleterious	0.950	Possibly Damaging	0.419	Benign	2.01	Pathogenic	0.00	Affected	4.32	2	-3	-3	5.4	16.04
c.3197C>T	P1066L	Likely Benign		Likely Benign		1	6-33443749-C-T	14	8.71e-6	-5.478	Likely Benign	0.092	Likely Benign	Likely Benign	0.173	Likely Benign										-3.68	Deleterious	0.996	Probably Damaging	0.903	Possibly Damaging	2.72	Benign	0.00	Affected	4.32	2	-3	-3	5.4	16.04
c.3328A>G	S1110G	Likely Benign		Likely Benign		1				-4.674	Likely Benign	0.079	Likely Benign	Likely Benign	0.035	Likely Benign										-2.26	Neutral	0.036	Benign	0.026	Benign	2.19	Pathogenic	0.08	Tolerated	4.32	2	1	0	0.4	-30.03
c.3395C>A	S1132Y	Likely Benign		Likely Benign		1				-5.894	Likely Benign	0.392	Ambiguous	Likely Benign	0.401	Likely Benign										-1.76	Neutral	0.500	Possibly Damaging	0.208	Benign	5.40	Benign	0.09	Tolerated	4.32	4	-3	-2	-0.5	76.10
c.3424T>C	S1142P	Likely Benign		Likely Benign		1	6-33444459-T-C	1	6.20e-7	-2.713	Likely Benign	0.222	Likely Benign	Likely Benign	0.107	Likely Benign										-2.19	Neutral	0.918	Possibly Damaging	0.761	Possibly Damaging	2.64	Benign	0.00	Affected	4.32	4	-1	1	-0.8	10.04
c.3567G>C	E1189D	Likely Benign	Coiled-coil	Likely Benign		1	6-33444602-G-C	3	1.86e-6	-3.582	Likely Benign	0.461	Ambiguous	Likely Benign	0.359	Likely Benign										-1.42	Neutral	0.992	Probably Damaging	0.989	Probably Damaging	5.30	Benign	0.25	Tolerated	3.82	4	3	2	0.0	-14.03
c.3632T>A	M1211K	Likely Pathogenic	Coiled-coil	Likely Benign		1				-9.013	Likely Pathogenic	0.662	Likely Pathogenic	Likely Benign	0.595	Likely Pathogenic										-2.95	Deleterious	0.987	Probably Damaging	0.979	Probably Damaging	5.59	Benign	0.01	Affected	3.77	5	0	-1	-5.8	-3.02
c.3821G>A	R1274H			Likely Benign		1	6-33447869-G-A	4	2.58e-6	-5.259	Likely Benign	0.256	Likely Benign	Likely Benign	0.149	Likely Benign										-3.20	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	2.49	Pathogenic	0.01	Affected	3.77	5	0	2	1.3	-19.05
c.3824G>T	R1275L			Likely Benign		1	6-33447872-G-T	1	6.45e-7	-6.052	Likely Benign	0.446	Ambiguous	Likely Benign	0.117	Likely Benign										-4.04	Deleterious	0.800	Possibly Damaging	0.277	Benign	2.55	Benign	0.01	Affected	3.77	5	-3	-2	8.3	-43.03
c.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.3970C>T	P1324S	Likely Benign		Likely Benign		1	6-33451844-C-T	5	3.26e-6	-5.451	Likely Benign	0.068	Likely Benign	Likely Benign	0.049	Likely Benign										0.35	Neutral	0.225	Benign	0.092	Benign	4.33	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
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.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.937G>A	E313K (3D Viewer)	Likely Pathogenic	C2	Likely Benign		1				-12.902	Likely Pathogenic	0.959	Likely Pathogenic	Likely Pathogenic	0.575	Likely Pathogenic	0.64	Ambiguous	0.6	1.40	Ambiguous	1.02	Ambiguous	0.75	Ambiguous	-3.31	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	1.90	Pathogenic	0.02	Affected			0	1	-0.4	-0.94
c.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.1352T>C	L451P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-14.549	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.750	Likely Pathogenic	6.92	Destabilizing	0.2	8.57	Destabilizing	7.75	Destabilizing	2.58	Destabilizing	-6.81	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.43	Pathogenic	0.00	Affected	3.37	34	-3	-3	-5.4	-16.04
c.1205T>G	L402R (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-13.800	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.522	Likely Pathogenic	4.10	Destabilizing	0.2	3.82	Destabilizing	3.96	Destabilizing	2.24	Destabilizing	-4.69	Deleterious	0.967	Probably Damaging	0.459	Possibly Damaging	3.69	Benign	0.00	Affected	3.38	28	-3	-2	-8.3	43.03	259.5	-55.4	0.0	0.0	1.4	0.0	X		X				X	Potentially Pathogenic	The iso-butyl side chain of Leu402, located in an anti-parallel β sheet strand (res. Ala399-Ile411), packs with residues inside the hydrophobic core of the C2 domain (e.g., Ile268, Ala404, Leu266, Val400). In the variant simulations, the positively charged guanidinium group of the Arg402 side chain is not suitable for the hydrophobic niche. Consequently, the side chain moves outward from the hydrophobic C2 domain core and stacks with the phenol ring of Tyr363 or forms H-bonds with the carboxamide group of the Gln361 side chain in the β sheet strand (res. Thr359-Tyr364). This movement induces extensive negative effects on the C2 domain structure.
c.1493T>G	M498R (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-8.812	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.869	Likely Pathogenic	3.85	Destabilizing	0.2	2.35	Destabilizing	3.10	Destabilizing	1.76	Destabilizing	-4.53	Deleterious	0.464	Possibly Damaging	0.120	Benign	-1.36	Pathogenic	0.00	Affected			0	-1	-6.4	24.99
c.1513T>C	Y505H (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-11.383	Likely Pathogenic	0.982	Likely Pathogenic	Likely Pathogenic	0.646	Likely Pathogenic	2.91	Destabilizing	0.1	2.88	Destabilizing	2.90	Destabilizing	1.60	Destabilizing	-4.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.64	Benign	0.00	Affected	3.37	35	2	0	-1.9	-26.03
c.1259T>C	F420S (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-13.231	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.544	Likely Pathogenic	5.34	Destabilizing	0.1	5.73	Destabilizing	5.54	Destabilizing	2.14	Destabilizing	-7.43	Deleterious	0.998	Probably Damaging	0.938	Probably Damaging	3.09	Benign	0.00	Affected	3.37	29	-3	-2	-3.6	-60.10	213.3	57.8	0.0	0.0	-0.4	0.1							X	Potentially Pathogenic	In the WT, the phenyl ring of the Phe420 side chain, located on an α helix (res. Met414-Glu436), packs against hydrophobic residues in the interhelix area of the GAP domain (e.g., Leu689, Leu714, Leu717, Leu718). Although no large-scale adverse effects are seen in the variant simulations, the polar hydroxyl group of Ser420 is not suitable for the hydrophobic inter-helix space. Thus, the residue swap could affect protein folding. In theory, the introduced hydroxyl group could also lower the α helix integrity by H-bonding with the backbone atoms of neighboring residues in the same α helix. However, no such effect is seen in the variant simulations.
c.1513T>G	Y505D (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-14.078	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.718	Likely Pathogenic	4.98	Destabilizing	0.1	4.72	Destabilizing	4.85	Destabilizing	2.49	Destabilizing	-9.95	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.60	Benign	0.00	Affected	3.37	35	-3	-4	-2.2	-48.09
c.1292T>C	L431P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-14.222	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.659	Likely Pathogenic	6.78	Destabilizing	0.3	11.59	Destabilizing	9.19	Destabilizing	2.29	Destabilizing	-6.39	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.91	Benign	0.05	Affected	3.37	29	-3	-3	-5.4	-16.04	222.4	62.8	0.1	0.0	0.1	0.0	X							Potentially Pathogenic	The iso-butyl side chain of Leu431, located in an α helix (res. Met414-Glu436), packs against other hydrophobic residues in an interhelix space (e.g., Val434, Leu435, Leu696, Leu711) in the WT simulations. While the backbone amide group of Leu431 forms an H-bond with the carbonyl group of His427, the cyclic five-membered pyrrolidine ring of Pro431, lacking the necessary amide group, cannot do the same. Thus, although the cyclic five-membered pyrrolidine ring of Pro431 packs almost as favorably as the side chain of Leu431 in the hydrophobic niche, the residue swap causes the α helix to partially unfold in the variant simulations.
c.1394T>C	L465P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-14.824	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.778	Likely Pathogenic	7.18	Destabilizing	0.3	10.85	Destabilizing	9.02	Destabilizing	2.73	Destabilizing	-6.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.29	Pathogenic	0.00	Affected	3.37	34	-3	-3	-5.4	-16.04	211.1	65.9	0.1	0.0	-0.2	0.0	X							Potentially Pathogenic	The iso-butyl side chain of Leu465, located in the middle of an α helix (res. Ala461–Phe476), packs with hydrophobic residues (e.g., Phe464, Met468, Tyr497, Ile494) in an inter-helix space formed with two other α helices (res. Ala461–Phe476 and res. Thr488-Gly502). In the variant simulations, the cyclic five-membered pyrrolidine ring of Pro465 is not as optimal as the side chain of Leu465 for filling the three α helix hydrophobic niche. Although the residue swap does not cause a large-scale conformational shift during the simulations, the H-bond between the backbone amide group of Leu465 and the backbone carbonyl group of Ala461 is lost. This, in turn, breaks the continuity of the α helix secondary structure element.
c.1403T>A	M468K (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-16.982	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.828	Likely Pathogenic	3.21	Destabilizing	0.1	3.30	Destabilizing	3.26	Destabilizing	2.57	Destabilizing	-4.61	Deleterious	0.878	Possibly Damaging	0.922	Probably Damaging	-1.34	Pathogenic	0.04	Affected	3.37	31	0	-1	-5.8	-3.02	188.7	69.3	0.0	0.0	-0.1	0.2			X				X	Potentially Pathogenic	The thioether group of Met468, located in the middle of an α helix (res. Ala461–Phe476), interacts with hydrophobic residues (e.g., Phe464, Leu465, Leu489) in an inter-helix space formed by two other α helices (res. Ala461–Phe476, res. Thr488–Gly502). In the variant simulations, the positively charged side chain of Lys468 rotates outward to escape the hydrophobic niche, forming an H-bond with the hydroxyl group of the Ser471 side chain and a salt bridge with the carboxylate group of the Glu472 side chain. This residue swap also disrupts the methionine-aromatic stacking with the phenyl ring of the Phe464 side chain. Although no large-scale structural changes are observed during the variant simulations, the importance of hydrophobic packing suggests that the effects could be more pronounced during protein folding.
c.1481T>G	I494R (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-15.758	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.911	Likely Pathogenic	6.71	Destabilizing	0.3	3.40	Destabilizing	5.06	Destabilizing	2.19	Destabilizing	-6.43	Deleterious	0.999	Probably Damaging	0.957	Probably Damaging	-1.41	Pathogenic	0.00	Affected	3.37	35	-2	-3	-9.0	43.03	273.9	-59.8	0.0	0.0	0.0	0.1	X	X	X				X	Potentially Pathogenic	The sec-butyl side chain of Ile494, located in an α-helix (res. Leu489-Glu519), packs against hydrophobic residues (e.g., Phe484, Leu465, Trp572, Ala493, Met468) in an inter-helix space (res. Leu489-Glu519 and res. Ala461-Phe476). In the variant simulations, the bulkier and positively charged residue, Arg494, weakens the integrity of the opposing helix. Additionally, the bulkier Arg494 stacks with Phe484, causing the α-helices to move farther apart to accommodate it. This mutation could have substantial negative effects due to the fundamental role of hydrophobic packing, which is disrupted by Arg494 during protein folding.
c.1517T>C	L506P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-12.088	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.737	Likely Pathogenic	5.48	Destabilizing	0.7	10.19	Destabilizing	7.84	Destabilizing	2.50	Destabilizing	-6.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	1.55	Pathogenic	0.00	Affected	3.37	35	-3	-3	-5.4	-16.04	182.6	64.9	0.1	0.0	0.2	0.1	X							Potentially Pathogenic	Leu506 is located in the middle of an α-helix (res. Gly502-Tyr518) within the inter-helix space of two helices (res. Gly502-Tyr518 and res. Glu582-Met603). In the WT simulations, the iso-butyl side chain of Leu506 hydrophobically packs with residues in the inter-helix space (e.g., Ile510, Phe597, Leu598, Ala601). In the variant simulations, the cyclic five-membered pyrrolidine ring of Pro506 is not as optimal as Leu506 for hydrophobic packing with nearby residues. Additionally, Pro506 cannot maintain the hydrogen bond with the backbone oxygen of Gly502 as Leu506 does in the WT, which disrupts the secondary structure element.
c.1529T>G	I510S (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-11.661	Likely Pathogenic	0.955	Likely Pathogenic	Ambiguous	0.926	Likely Pathogenic	4.00	Destabilizing	0.1	3.78	Destabilizing	3.89	Destabilizing	2.34	Destabilizing	-4.63	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.44	Pathogenic	0.00	Affected	3.37	35	-1	-2	-5.3	-26.08	201.4	45.9	-0.4	0.2	0.0	0.3							X	Potentially Pathogenic	Ile510 is located in the middle of an α-helix (res. Gly502-Tyr518) within the inter-helix space of three helices (res. Gly502-Tyr518, Ala533-Val560, and res. Glu582-Met603). In the WT simulations, the sec-butyl side chain of Ile510 hydrophobically packs with other residues in the inter-helix space (e.g., Leu506, Leu610, Ile514, Ile602, Leu598). In the variant simulations, the hydroxyl group of Ser510 forms a hydrogen bond with the backbone atoms of Leu506 and Gly511 in the same α-helix, which could further weaken the α-helix integrity. This α-helix already shows weakness in the WT simulations due to Gly511. Although the simulations do not show large-scale effects, the residue swap could have a substantial impact due to the fundamental role of hydrophobic packing during protein folding.
c.1531G>A	G511R (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-11.327	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.416	Likely Benign	1.94	Ambiguous	0.3	1.32	Ambiguous	1.63	Ambiguous	0.94	Ambiguous	-7.72	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.26	Benign	0.06	Tolerated	3.37	35	-3	-2	-4.1	99.14	279.4	-159.9	0.0	0.0	0.7	0.1			X				X	Potentially Pathogenic	Gly511 is located in an α-helix (res. Gly502-Tyr518), facing hydrophobic residues in an inter-helix space (e.g., Leu610, Ile514) in the WT simulations. In contrast, in the variant simulations, the bulkier and positively charged guanidinium side chain of Arg511 forms a salt bridge with the carboxylate group of Glu217 or hydrogen bonds with the backbone carbonyl group of Leu610. Although the residue swap introduces a third positively charged residue in close vicinity (Arg511, Lys507, Arg515), the protein structure seems to remain stable in the variant simulations. Importantly, according to ClinVar, the residue swap alters the last nucleotide of an exon and is predicted to destroy the splice donor site, resulting in aberrant splicing and pathogenic status.	10.1016/j.ajhg.2020.11.011
c.1556A>C	E519A (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-8.557	Likely Pathogenic	0.904	Likely Pathogenic	Ambiguous	0.384	Likely Benign	-0.05	Likely Benign	0.0	0.55	Ambiguous	0.25	Likely Benign	0.00	Likely Benign	-5.23	Deleterious	0.999	Probably Damaging	0.998	Probably Damaging	3.33	Benign	0.10	Tolerated	3.37	35	0	-1	5.3	-58.04	162.4	83.5	-0.1	0.1	-0.2	0.0						X		Potentially Benign	Glu519 is located at the beginning of an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the carboxylate side chain of Glu519 does not make any specific interactions. Accordingly, the Ala residue swap does not show any negative structural effects in the variant simulations. However, it should be noted that Glu519 faces the missing part of the N-terminal in the model, and thus its potential role in maintaining the tertiary structure might be de-emphasized in the current model.
c.1652T>C	L551P (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-14.620	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.953	Likely Pathogenic	6.66	Destabilizing	0.1	6.58	Destabilizing	6.62	Destabilizing	2.66	Destabilizing	-4.70	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.60	Pathogenic	0.01	Affected	3.37	35	-3	-3	-5.4	-16.04	208.6	60.9	0.1	0.0	-0.3	0.0	X							Potentially Pathogenic	L551 is located on an α-helix (res. Ala533-Val560). The iso-butyl side chain of Leu551 hydrophobically packs with nearby hydrophobic residues such as Cys547, Phe652, Leu633, and Ile630 in the inter-helix space. In the variant simulations, the pyrrolidine side chain of Pro551 is not as optimal as leucine for hydrophobic packing with the nearby residues. Moreover, Pro551 lacks the amide group, and thus, it cannot form a hydrogen bond with the backbone carbonyl group of Cys547, which disrupts the continuity of the secondary structure element.
c.1706T>C	F569S (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		2				-13.384	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.916	Likely Pathogenic	5.70	Destabilizing	0.1	5.38	Destabilizing	5.54	Destabilizing	2.45	Destabilizing	-7.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.32	Pathogenic	0.00	Affected	3.37	34	-3	-2	-3.6	-60.10	213.7	67.9	-0.1	0.0	-1.0	0.1							X	Potentially Pathogenic	Phe569 is located on an α-helix (res. Arg563-Glu578). In the WT simulations, the phenyl side chain of Phe569 packs with hydrophobic residues such as Trp572, Leu565, Ile589, Ile667, and Phe561, originating from three different α-helices (res. Ala533-Val560, res. Arg563-Glu578, and res. Ser641-Glu666). In the variant simulations, the acceptor/donor hydroxyl group of Ser569 forms hydrogen bonds with the carbonyl groups of Glu567 and Lys566 on the same α-helix, which could affect the α-helix integrity, although this is not observed in the simulations. While the simulations do not show large-scale effects, the residue swap could have a substantial impact on the protein structure due to the fundamental role of hydrophobic packing during protein folding.
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.1718G>A	R573Q (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-9.900	Likely Pathogenic	0.923	Likely Pathogenic	Ambiguous	0.733	Likely Pathogenic	2.28	Destabilizing	0.8	1.94	Ambiguous	2.11	Destabilizing	1.08	Destabilizing	-3.16	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.31	Pathogenic	0.12	Tolerated	3.37	35	1	1	1.0	-28.06	230.1	49.9	0.0	0.0	-0.6	0.0	X	X						Potentially Pathogenic	The guanidinium group of Arg573, located in an α-helix (res. Arg563-Glu578), forms a salt bridge with the carboxylate groups of Glu582 and/or Asp586 from a nearby α-helix (res. Glu582-Met603) in the WT simulations. Additionally, the Arg573 side chain stacks planarly with the aromatic phenol ring of Tyr665 and hydrogen bonds with the hydroxyl group of Ser668 from another α-helix (res. Ser641-Ser668). In the variant simulations, although the carboxamide group of the Gln573 side chain can hydrogen bond with the carboxylate group of Glu582 or the hydroxyl group of Ser668, these interactions are not as coordinated, stable, or strong as those of the positively charged Arg573. Consequently, the integrity of the opposing α-helix end (res. Glu582-Met603) is weakened. Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.
c.1718G>T	R573L (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-13.120	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.833	Likely Pathogenic	1.30	Ambiguous	0.6	1.11	Ambiguous	1.21	Ambiguous	0.80	Ambiguous	-5.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.41	Pathogenic	0.01	Affected	3.37	35	-3	-2	8.3	-43.03	237.4	60.7	0.0	0.0	-0.7	0.3	X	X						Potentially Pathogenic	The guanidinium group of Arg573, located in an α-helix (res. Arg563-Glu578), forms a salt bridge with the carboxylate groups of Glu582 and/or Asp586 from a nearby α-helix (res. Glu582-Met603) in the WT simulations. Additionally, the Arg573 side chain stacks planarly with the aromatic phenol ring of Tyr665 and hydrogen bonds with the hydroxyl group of Ser668 from another α-helix (res. Ser641-Ser668). In the variant simulations, the aliphatic iso-butyl group of the Leu573 side chain fails to establish any of these interactions, which, in turn, lowers the integrity of the opposing α-helix end (res. Glu582-Met603). Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.	10.1016/j.ajhg.2020.11.011
c.1763T>A	L588H (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-16.947	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.939	Likely Pathogenic	4.20	Destabilizing	0.2	3.69	Destabilizing	3.95	Destabilizing	2.26	Destabilizing	-6.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.42	Pathogenic	0.00	Affected	3.38	34	-2	-3	-7.0	23.98	214.3	20.9	0.0	0.0	0.0	0.2	X	X					X	Potentially Pathogenic	The isobutyl group of the Leu588 side chain, located in an α helix (res. Glu582-Met603), packs against hydrophobic residues in the inter-helix hydrophobic space (e.g., Ile584, Trp572, Phe484, Met470, Val473, Ile483).In the variant simulations, the imidazole ring of His588 is aromatic but contains polar delta and epsilon nitrogen atoms that are not suited for the hydrophobic niche. The protonated epsilon nitrogen forms a hydrogen bond with the backbone carbonyl group of Ala469, which can disrupt the continuity of the opposing α helix (res. Phe476-Lys460).While the residue swap could affect the tertiary assembly and the underlying protein folding process, it is difficult to determine if the mutation would be tolerated based solely on the variant simulations.
c.1925A>C	K642T (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-12.823	Likely Pathogenic	0.948	Likely Pathogenic	Ambiguous	0.484	Likely Benign	0.53	Ambiguous	0.1	0.30	Likely Benign	0.42	Likely Benign	0.28	Likely Benign	-5.88	Deleterious	0.872	Possibly Damaging	0.839	Possibly Damaging	2.86	Benign	0.00	Affected	3.37	31	0	-1	3.2	-27.07	213.5	-8.7	-0.3	0.4	0.3	0.2				X				Uncertain	The amino side chain of Lys642, located on the surface of an α helix (res. Ser641-Glu666), is not involved in any interactions in the WT simulations. In the variant simulations, the shorter side chain of Thr642 forms hydrogen bonds with Glu643 and Thr640 on the same α helix.Regardless, Lys642 is positioned directly at the GAP-Ras interface, and in the SynGAP-Ras WT simulations, its amino side chain forms salt bridges with the carboxylate groups of Ras residues Asp33 and Asp38. The shorter Thr642 is more likely to prefer hydrogen bonding with Glu643 and Thr640 on the same α helix, even in the Ras complex. Thus, the effect of the residue swap on the complex formation with the GTPase cannot be explored using solvent-only simulations.
c.2003C>T	S668F (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-15.047	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.643	Likely Pathogenic	16.72	Destabilizing	5.0	11.07	Destabilizing	13.90	Destabilizing	0.00	Likely Benign	-5.98	Deleterious	0.999	Probably Damaging	0.935	Probably Damaging	3.18	Benign	0.00	Affected	3.38	28	-3	-2	3.6	60.10	250.9	-59.6	-0.1	0.1	0.0	0.1		X	X				X	Potentially Pathogenic	In the WT simulations, the hydroxyl side chain of Ser668, located on an α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), forms hydrogen bonds with the backbone carbonyl groups of Leu664, Tyr665, and Glu666, as well as the guanidinium group of Arg573 on a nearby α-helix (res. Arg563-Glu578). In the variant simulations, the side chain of Phe668 cannot maintain the same hydrogen-bond network. Due to its larger size, it moves away to avoid steric hindrance. In the WT simulations, a network of hydrogen bonds between several residues (e.g., Asn669, Lys566, and Glu666) keeps both α-helices and the proceeding loop (res. Asn669-Asp684) tightly connected, but this setup is not present in the variant simulations. Additionally, in the variant simulations, the side chain of Arg573 shifts to form a more stable salt bridge with the carboxylate group of Glu582 instead of hydrogen bonding with Ser668 as in the WT simulations.
c.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.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.2525C>A	S842Y	Likely Pathogenic		Likely Pathogenic		1				-16.124	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.191	Likely Benign										-4.28	Deleterious	0.944	Possibly Damaging	0.676	Possibly Damaging	1.97	Pathogenic	0.00	Affected	3.77	5	-3	-2	-0.5	76.10
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.2852A>G	H951R	Likely Benign		Likely Pathogenic		1				-4.964	Likely Benign	0.125	Likely Benign	Likely Benign	0.185	Likely Benign										-1.08	Neutral	0.048	Benign	0.029	Benign	5.46	Benign	0.24	Tolerated	3.77	5	2	0	-1.3	19.05
c.3151G>T	G1051C			Likely Pathogenic		1				-9.050	Likely Pathogenic	0.122	Likely Benign	Likely Benign	0.497	Likely Benign										-0.90	Neutral	0.971	Probably Damaging	0.750	Possibly Damaging	-0.74	Pathogenic	0.10	Tolerated	3.77	5	-3	-3	2.9	46.09
c.3196C>T	P1066S	Likely Benign		Likely Pathogenic		1				-4.746	Likely Benign	0.070	Likely Benign	Likely Benign	0.145	Likely Benign										-2.47	Neutral	0.972	Probably Damaging	0.850	Possibly Damaging	2.74	Benign	0.00	Affected	4.32	2	1	-1	0.8	-10.04
c.3794G>C	R1265T	Likely Pathogenic	Coiled-coil	Likely Pathogenic		1				-10.129	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.529	Likely Pathogenic										-4.97	Deleterious	0.997	Probably Damaging	0.994	Probably Damaging	2.29	Pathogenic	0.00	Affected	3.77	5	-1	-1	3.8	-55.08
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.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.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.851T>C	L284P	Likely Pathogenic	C2	Likely Pathogenic		1				-15.588	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.794	Likely Pathogenic	5.83	Destabilizing	0.2	5.81	Destabilizing	5.82	Destabilizing	1.89	Destabilizing	-6.17	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	1.64	Pathogenic	0.00	Affected			-3	-3	-5.4	-16.04
c.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.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.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.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.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.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.1685C>T	P562L (3D Viewer)	Likely Pathogenic	GAP	Pathogenic/Likely path.		10	6-33440737-C-T			-13.438	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.829	Likely Pathogenic	3.54	Destabilizing	0.8	0.17	Likely Benign	1.86	Ambiguous	-0.14	Likely Benign	-9.95	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	0.58	Pathogenic	0.00	Affected	3.37	35	-3	-3	5.4	16.04	228.8	-68.5	-0.1	0.0	0.1	0.2							X	Potentially Pathogenic	Pro562 is located on an α-α loop between two α-helices (res. Ala533-Val560 and res. Arg563-Glu578). The cyclic pyrrolidine side chain of Pro562 hydrophobically packs with other residues in the inter-helix space, such as Leu565, Ile501, and Phe561. In the variant simulations, Leu562 packs more favorably with the nearby hydrophobic residues, and the backbone amide group of Leu562 (absent in proline) does not form any intra-protein hydrogen bonds. However, prolines are well-suited for unstructured regions like loops, and thus, Pro562 in the WT is necessary at the end of the helix to induce a tight turn during folding. Although no negative structural effects are observed during the simulations, the residue swap could potentially cause extensive damage to the protein structure during folding.	10.1016/j.ajhg.2020.11.011
c.1898T>C	L633P (3D Viewer)	Likely Pathogenic	GAP	Pathogenic/Likely path.		2				-15.669	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.693	Likely Pathogenic	6.60	Destabilizing	0.2	10.15	Destabilizing	8.38	Destabilizing	2.42	Destabilizing	-6.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.70	Benign	0.00	Affected	3.37	34	-3	-3	-5.4	-16.04	193.2	65.1	0.0	0.0	0.1	0.0	X							Potentially Pathogenic	The iso-butyl side chain of Leu633, located in the middle of an α helix (res. Glu617-Asn635), packs hydrophobically with nearby residues (e.g., Leu653, Val629, Leu551) in the WT simulations.In the variant simulations, the pyrrolidine side chain of Pro633 is not as optimal for hydrophobic packing as Leu633 in the WT. Additionally, proline lacks a free backbone amide group, so Pro633 cannot form a hydrogen bond with the backbone carbonyl group of Val629, which disrupts the continuity of the secondary structure element.
c.509G>A	R170Q			Pathogenic/Likely path.		6				-9.021	Likely Pathogenic	0.798	Likely Pathogenic	Ambiguous	0.221	Likely Benign										-2.31	Neutral	0.947	Possibly Damaging	0.342	Benign	3.91	Benign	0.00	Affected	3.74	4	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.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.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.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.1499T>C	L500P (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-15.898	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.894	Likely Pathogenic	5.91	Destabilizing	0.3	8.90	Destabilizing	7.41	Destabilizing	1.92	Destabilizing	-6.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.37	Pathogenic	0.01	Affected	3.37	35	-3	-3	-5.4	-16.04
c.1531G>C	G511R (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-11.327	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.415	Likely Benign	1.94	Ambiguous	0.3	1.32	Ambiguous	1.63	Ambiguous	0.94	Ambiguous	-7.72	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.26	Benign	0.06	Tolerated	3.37	35	-3	-2	-4.1	99.14	279.4	-159.9	0.0	0.0	0.7	0.1			X				X	Potentially Pathogenic	Gly511 is located in an α-helix (res. Gly502-Tyr518), facing hydrophobic residues in an inter-helix space (e.g., Leu610, Ile514) in the WT simulations. In contrast, in the variant simulations, the bulkier and positively charged guanidinium side chain of Arg511 forms a salt bridge with the carboxylate group of Glu217 or hydrogen bonds with the backbone carbonyl group of Leu610. Although the residue swap introduces a third positively charged residue in close vicinity (Arg511, Lys507, Arg515), the protein structure seems to remain stable in the variant simulations. Importantly, according to ClinVar, the residue swap alters the last nucleotide of an exon and is predicted to destroy the splice donor site, resulting in aberrant splicing and pathogenic status.	10.1016/j.ajhg.2020.11.011
c.1640G>A	C547Y (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-15.871	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.874	Likely Pathogenic	8.53	Destabilizing	1.8	6.20	Destabilizing	7.37	Destabilizing	0.62	Ambiguous	-10.57	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.33	Pathogenic	0.06	Tolerated	3.37	35	0	-2	-3.8	60.04	280.1	-54.8	0.0	0.0	0.0	0.0		X	X				X	Potentially Pathogenic	Cys547 is located in an α-helix (res. Ala533-Val560). The thiol side chain of Cys547 is situated in a hydrophobic inter-helix space, where it packs hydrophobically with other residues such as Ile626, Leu551, and Phe652. Additionally, the thiol side chain of Cys weakly hydrogen bonds with the carbonyl group of Leu543 in the same α-helix. In the variant simulations, the bulkier phenol ring of Tyr547, with its polar hydroxyl group, is less suited for the hydrophobic space. Consequently, it moves outside and forms a hydrogen bond with the carbonyl group of Phe652 in the neighboring α-helix (res. Glu666-Asp644). This causes the two helices to slightly separate, negatively affecting the secondary structure integrity of the latter helix. These negative structural effects could be more pronounced during protein folding and are likely to be undermined in the MD simulations.
c.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.1715G>C	W572S (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-17.461	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.775	Likely Pathogenic	5.78	Destabilizing	0.2	3.37	Destabilizing	4.58	Destabilizing	1.79	Destabilizing	-12.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.24	Pathogenic	0.01	Affected	3.37	35	-2	-3	0.1	-99.14	235.1	76.6	0.0	0.0	-0.4	0.1							X	Potentially Pathogenic	The introduced residue Ser572, located in an α-helix (res. Arg563-Glu578), is considerably smaller than the tryptophan it replaced. The indole ring of the Trp572 side chain lies in a hydrophobic inter-helix space, where it makes extensive hydrophobic interactions with nearby residues such as Met470, Phe569, Leu588, and Ile589. In the variant simulations, all these favorable packing interactions are completely removed, as the introduced residue Ser572 is too hydrophilic or small to fill the hydrophobic niche occupied by the indole ring. Moreover, the hydroxyl group of Ser572 forms hydrogen bonds with the carbonyl groups of Glu567 and Val568 within the same α-helix, potentially lowering its integrity. Overall, the residue swap is highly likely to cause critical protein folding problems that are underestimated based on the effects seen in the variant simulations.
c.2485G>A	E829K	Likely Pathogenic		Pathogenic		1				-7.527	In-Between	0.807	Likely Pathogenic	Ambiguous	0.194	Likely Benign										-2.65	Deleterious	0.994	Probably Damaging	0.900	Possibly Damaging	2.27	Pathogenic	0.00	Affected	3.77	5	0	1	-0.4	-0.94
c.484C>T	R162C			Pathogenic		2				-8.157	Likely Pathogenic	0.787	Likely Pathogenic	Ambiguous	0.150	Likely Benign										-2.05	Neutral	0.988	Probably Damaging	0.513	Possibly Damaging	4.00	Benign	0.11	Tolerated	3.74	4	-4	-3	7.0	-53.05
c.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.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.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.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.113C>T	P38L	Likely Benign		Conflicting		4	6-33423522-C-T	8	4.96e-6	-2.469	Likely Benign	0.197	Likely Benign	Likely Benign	0.141	Likely Benign										-2.56	Deleterious	0.983	Probably Damaging	0.931	Probably Damaging	4.02	Benign	0.00	Affected	4.32	1	-3	-3	5.4	16.04
c.121C>T	R41C	Likely Benign		Conflicting		3	6-33423530-C-T	7	4.34e-6	-4.745	Likely Benign	0.207	Likely Benign	Likely Benign	0.093	Likely Benign										-1.10	Neutral	0.976	Probably Damaging	0.919	Probably Damaging	4.13	Benign	0.00	Affected	4.32	1	-4	-3	7.0	-53.05
c.1447A>G	I483V (3D Viewer)		GAP	Conflicting		2				-10.121	Likely Pathogenic	0.523	Ambiguous	Likely Benign	0.228	Likely Benign	1.00	Ambiguous	0.0	0.27	Likely Benign	0.64	Ambiguous	1.02	Destabilizing	-0.86	Neutral	0.914	Possibly Damaging	0.921	Probably Damaging	3.23	Benign	0.03	Affected	3.37	32	3	4	-0.3	-14.03
c.1213C>T	R405C (3D Viewer)	Likely Pathogenic	C2	Conflicting		2	6-33438118-C-T	6	3.72e-6	-9.206	Likely Pathogenic	0.713	Likely Pathogenic	Likely Benign	0.427	Likely Benign	0.72	Ambiguous	0.1	1.51	Ambiguous	1.12	Ambiguous	1.21	Destabilizing	-7.27	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.61	Benign	0.02	Affected	3.38	28	-4	-3	7.0	-53.05	221.3	82.6	-0.1	0.0	-0.2	0.3	X	X						Potentially Pathogenic	The guanidinium group of Arg405, located in an anti-parallel β sheet strand of the C2 domain (res. Ala399-Ile411), forms a salt bridge with the carboxylate group of the Glu446 side chain from an opposing α helix (res. Val441-Ser457) in the GAP domain. The positively charged Arg405 side chain also stacks with the aromatic ring of the Phe358 side chain from a loop preceding the β strand (res. Thr359-Thr366), which could assist in maintaining the anti-parallel strand arrangement.In the variant simulations, the thiol-containing side chain of Cys405 is neutral and smaller compared to the arginine side chain. The lack of Arg405-Phe358 stacking affects the loop structure, causing it to assume a β strand form—an effect that could be exacerbated during protein folding. Moreover, the inability of Cys405 to form a salt bridge with Glu446 could affect the tertiary structure assembly, although this is not apparent based on the variant simulations.
c.1474A>G	K492E (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-16.175	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.510	Likely Pathogenic	1.53	Ambiguous	0.1	1.90	Ambiguous	1.72	Ambiguous	1.42	Destabilizing	-3.98	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.99	Benign	0.01	Affected	3.37	35	1	0	0.4	0.94
c.1214G>A	R405H (3D Viewer)	Likely Pathogenic	C2	Conflicting		2	6-33438119-G-A	4	2.48e-6	-9.081	Likely Pathogenic	0.706	Likely Pathogenic	Likely Benign	0.371	Likely Benign	2.79	Destabilizing	0.6	1.85	Ambiguous	2.32	Destabilizing	1.26	Destabilizing	-4.54	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	3.65	Benign	0.01	Affected	3.38	28	2	0	1.3	-19.05	214.0	102.2	-0.1	0.0	-0.7	0.1		X						Potentially Pathogenic	The guanidinium group of Arg405, located in an anti-parallel β sheet strand of the C2 domain (res. Pro398-Ile411), forms a salt bridge with the carboxylate group of the Glu446 side chain from an opposing α helix (res. Val441-Ser457) in the GAP domain. The positively charged Arg405 side chain also stacks with the aromatic ring of the Phe358 side chain from a loop preceding the β strand (res. Thr359-Thr366), which could assist in maintaining the anti-parallel strand arrangement.In the variant simulations, the imidazole ring of His405 does not stack with the aromatic ring of Phe358 nor form any lasting H-bonds with the loop residues. The imidazole ring of His405 (neutral and epsilon protonated in the simulations) is unable to form a salt bridge with Glu446, which could affect the tertiary structure assembly, although this is not apparent based on the variant simulations.
c.1285C>T	R429W (3D Viewer)		GAP	Conflicting		5	6-33438190-C-T	65	4.03e-5	-10.666	Likely Pathogenic	0.500	Ambiguous	Likely Benign	0.282	Likely Benign	0.31	Likely Benign	0.1	-0.13	Likely Benign	0.09	Likely Benign	0.52	Ambiguous	-3.19	Deleterious	1.000	Probably Damaging	0.990	Probably Damaging	3.41	Benign	0.03	Affected	3.38	25	2	-3	3.6	30.03	252.3	45.5	0.0	0.0	0.2	0.1		X						Potentially Pathogenic	The guanidinium group of Arg429, located in an α helix (res. Met414-Glu436), either forms a salt bridge with the carboxylate group of an acidic residue (Asp474, Asp467) or a H-bond with the hydroxyl group of Ser471 in an opposing α helix (res. Ala461-Phe476). In the variant simulations, the indole ring of the Trp429 side chain cannot form ionic interactions with the acidic residues. Although it forms a H-bond with Ser471, the bonding is not as strong as that of arginine. The residue swap could affect the tertiary structure assembly during folding; however, no large-scale negative effects were seen during the simulations.
c.1312G>A	A438T (3D Viewer)	Likely Benign	GAP	Conflicting		3	6-33438217-G-A	16	9.91e-6	-5.339	Likely Benign	0.085	Likely Benign	Likely Benign	0.021	Likely Benign	0.21	Likely Benign	0.0	-0.07	Likely Benign	0.07	Likely Benign	0.36	Likely Benign	-0.81	Neutral	0.300	Benign	0.011	Benign	4.18	Benign	0.14	Tolerated	3.38	26	1	0	-2.5	30.03	214.2	-42.7	-0.3	0.1	-0.4	0.1						X		Potentially Benign	The methyl group of Ala438, located in a four-residue loop connecting two α helices (res. Asn440-Thr458 and Pro413-Glu436), packs against hydrophobic residues from a nearby α helix or loop residues (e.g., Leu703, Val699). In the variant simulations, the methyl group of Thr438 is able to establish similar hydrophobic packing. Moreover, the hydroxyl group also H-bonds with nearby residues, such as the carbonyl group of the neighboring loop residue Pro437. Accordingly, the residue swap does not generate an apparent negative effect on the protein structure based on the simulations.
c.1322T>C	V441A (3D Viewer)		GAP	Conflicting		2	6-33438227-T-C	3	1.86e-6	-9.439	Likely Pathogenic	0.359	Ambiguous	Likely Benign	0.053	Likely Benign	-0.14	Likely Benign	0.0	0.33	Likely Benign	0.10	Likely Benign	0.95	Ambiguous	-2.92	Deleterious	0.513	Possibly Damaging	0.214	Benign	3.44	Benign	0.93	Tolerated	3.37	29	0	0	-2.4	-28.05	195.0	44.6	0.0	0.1	0.5	0.0				X		X		Uncertain	The iso-propyl side chain of Val441, located on the outer surface of an α helix (res. Asn440-Thr458), does not interact with other residues in the WT simulations. In the variant simulations, the methyl side chain of Ala441 is similarly hydrophobic and does not form any interactions on the outer helix surface. Although the residue swap does not negatively affect the protein structure based on the simulations, it is noteworthy that the residue faces the RasGTPase interface. Thus, the effect of the residue swap on the SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.1726T>C	C576R (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-14.886	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.579	Likely Pathogenic	7.20	Destabilizing	1.0	4.09	Destabilizing	5.65	Destabilizing	1.64	Destabilizing	-10.88	Deleterious	0.999	Probably Damaging	0.996	Probably Damaging	3.38	Benign	0.00	Affected	3.37	35	-3	-4	-7.0	53.05
c.1466T>C	L489P (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-13.520	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.939	Likely Pathogenic	2.50	Destabilizing	0.1	4.69	Destabilizing	3.60	Destabilizing	1.73	Destabilizing	-6.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.56	Pathogenic	0.00	Affected	3.37	35	-3	-3	-5.4	-16.04	209.9	61.9	0.1	0.0	0.6	0.1		X						Potentially Pathogenic	The iso-butyl side chain of Leu489, located in the α-helix (res. Leu489-Glu519) within an inter-helix space of four helices (res. Ala461-Phe476, res. Val441-Ser457, and res. Met414-Glu436), packs with hydrophobic residues (e.g., Cys432, Ala448, Lys444, Ala493, Val447, Met468). In the variant simulations, Pro489 is located near the beginning of the α-helix, so the residue swap with Leu489 does not affect the continuity of the secondary structure element. However, the side chain of proline is not as optimal as that of leucine for maintaining hydrophobic packing with nearby residues (e.g., Ala448, Lys444). Additionally, the consistently maintained hydrogen bond interaction between the backbone amide group of Leu489 and the carbonyl of Glu436 is lost due to the residue swap, potentially affecting the tertiary structure integrity.