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.1025A>C	Y342S (3D Viewer)	Likely Pathogenic	C2	Uncertain		2				-7.996	In-Between	0.925	Likely Pathogenic	Ambiguous	0.407	Likely Benign	3.03	Destabilizing	0.1	2.87	Destabilizing	2.95	Destabilizing	0.93	Ambiguous	-6.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	1.75	Pathogenic	0.04	Affected	3.37	25	-3	-2	0.5	-76.10	200.1	77.8	0.0	0.0	-0.2	0.1											Potentially Pathogenic	The phenol ring of Tyr342, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), faces outward in the C2 domain. In the WT simulations, the phenol ring of Tyr342 contributes to a triple tyrosine stack (Tyr342, Tyr328, and Tyr281) that links together three anti-parallel β sheet strands. Additionally, it shields Gly344 from the solvent, reducing its exposure and providing stability for the β-sandwich. This motif also contributes to a twist formation in the β sheet.In the variant simulations, the Ser342 side chain cannot participate in the stack formation. Instead, the hydroxyl group of the Ser342 side chain forms a hydrogen bond with the imidazole ring of His326 in a neighboring β strand (res. Ala322-Asp330). This disrupts the formation of a hydrogen bond between His326 and the carboxylate group of the Glu283 side chain from another β strand (res. Arg279-Cys285). Although these changes in surface interactions could weaken the characteristic twist that strengthens the β sheet fold, no major structural effects are observed in the variant simulations. The residue swap could also affect the SynGAP-membrane association, as the hydroxyl group of Ser342 could form hydrogen bonds with membrane-facing loop residues. However, this phenomenon cannot be addressed using solvent-only simulations.
c.1058T>C	L353P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-7.913	In-Between	0.936	Likely Pathogenic	Ambiguous	0.464	Likely Benign	4.63	Destabilizing	0.1	10.19	Destabilizing	7.41	Destabilizing	2.17	Destabilizing	-3.70	Deleterious	0.947	Possibly Damaging	0.454	Possibly Damaging	1.29	Pathogenic	0.02	Affected	3.37	25	-3	-3	-5.4	-16.04
c.1082A>C	Q361P (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-13.280	Likely Pathogenic	0.956	Likely Pathogenic	Likely Pathogenic	0.482	Likely Benign	3.12	Destabilizing	0.0	3.45	Destabilizing	3.29	Destabilizing	0.38	Likely Benign	-3.03	Deleterious	0.996	Probably Damaging	0.979	Probably Damaging	1.63	Pathogenic	0.05	Affected	3.37	25	-1	0	1.9	-31.01
c.1118G>A	G373E (3D Viewer)		C2	Uncertain		1				-7.281	In-Between	0.569	Likely Pathogenic	Likely Benign	0.420	Likely Benign	4.13	Destabilizing	3.2	0.52	Ambiguous	2.33	Destabilizing	-0.02	Likely Benign	-0.69	Neutral	0.001	Benign	0.000	Benign	3.90	Benign	0.01	Affected			0	-2	-3.1	72.06
c.1030G>A	G344S (3D Viewer)	Likely Pathogenic	C2	Pathogenic		5				-11.254	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.790	Likely Pathogenic	9.02	Destabilizing	0.7	6.08	Destabilizing	7.55	Destabilizing	0.98	Ambiguous	-5.28	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-0.45	Pathogenic	0.04	Affected	3.37	25	1	0	-0.4	30.03	217.3	-51.7	0.0	0.1	0.2	0.1			X				X	Potentially Pathogenic	Because Gly344 lacks a proper side chain, it allows the anti-parallel β sheet strand (res. Gly341-Pro349) to have a slight twist. Within a β strand, side chains normally alternate between outward and inward positions, but glycine is an exception as it allows the alternating pattern to skip a residue. Introducing serine or any other residue with a side chain at position 344 prevents this unique skip in the alternating pattern, causing structural strain or likely preventing correct folding altogether. Additionally, Tyr342 shields Gly344 from the solvent, contributing to twist formation in the β sheet and stabilizing the β-strand.In the variant simulations, the side chain of Ser344 assumes the inward position. However, the hydrophobic niche formed by multiple C2 domain residues (e.g., Val365, Val343, Leu327) is not accommodating for its hydroxyl group. The outward position, not seen in the simulations, would be equally disadvantageous due to the presence of hydrophobic residues on that side as well (e.g., Leu345, Tyr342). Serine is also not well-suited for twist formation, as it tends to suppress twisting and bending in β sheets. At this position, the hydroxyl group of Ser344 could also form hydrogen bonds with the backbone atoms of the Gly-rich Ω loop in the C2 domain (e.g., Thr366, Leu367, Gly378; res. Pro364-Pro398), potentially adversely affecting membrane-loop dynamics and ultimately compromising the stability of the SynGAP-membrane association.
c.1042G>A	V348M (3D Viewer)		C2	Uncertain		1				-7.076	In-Between	0.546	Ambiguous	Likely Benign	0.191	Likely Benign	-1.19	Ambiguous	0.1	0.72	Ambiguous	-0.24	Likely Benign	0.76	Ambiguous	-1.62	Neutral	0.966	Probably Damaging	0.564	Possibly Damaging	1.58	Pathogenic	0.03	Affected	3.37	25	2	1	-2.3	32.06	253.8	-47.4	-0.3	0.1	0.2	0.1						X		Potentially Benign	The iso-propyl side chain of Val348, located in an anti-parallel β sheet strand (res. Gly341-Pro349), packs against multiple hydrophobic C2 domain residues (e.g., Leu353, Leu323, Leu402). In the variant simulations, the thioether side chain of Met348 can form similar interactions as valine due to its comparable hydrophobic profile. In fact, the thioether group of methionine can even stack favorably with the phenol ring of Tyr363 in the anti-parallel β sheet strand (res. Ala399-Ile411). Overall, the residue swap does not appear to cause negative effects on the protein structure based on the simulations.
c.1045C>T	P349S (3D Viewer)		C2	Uncertain		1				-7.654	In-Between	0.217	Likely Benign	Likely Benign	0.277	Likely Benign	1.92	Ambiguous	0.1	2.28	Destabilizing	2.10	Destabilizing	0.87	Ambiguous	-6.13	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	1.66	Pathogenic	0.06	Tolerated	3.37	25	1	-1	0.8	-10.04	194.9	-18.1	-0.1	0.0	0.2	0.1	X						X	Potentially Pathogenic	The cyclic pyrrolidine side chain of Pro349, located at the end of an anti-parallel β sheet strand (res. Gly341-Pro349), allows the strand to end and make a tight turn before a short α helical section within a loop connecting to another β strand (res. Thr359-Pro364). In the variant simulations, the hydroxyl group of Ser349 forms a hydrogen bond with the backbone amide group of Ala351 in the short helical section. Conversely, the backbone amide group of Ser349 (absent in proline) does not form any intra-protein hydrogen bonds. However, the β strand end connects to the α helical section in a more stable and consistent manner compared to the WT. Although the residue swap does not cause major adverse effects on the protein structure in the simulations, it is possible that the tight turn at the β strand end could not be created during folding without the presence of proline.
c.1126G>T	G376C		C2	Uncertain		1				-7.686	In-Between	0.125	Likely Benign	Likely Benign	0.560	Likely Pathogenic	2.56	Destabilizing	0.5	0.22	Likely Benign	1.39	Ambiguous	0.16	Likely Benign	-1.15	Neutral	1.000	Probably Damaging	1.000	Probably Damaging	1.32	Pathogenic	0.01	Affected			-3	-3	2.9	46.09
c.1157G>A	G386E (3D Viewer)		C2	Uncertain		1	6-33438062-G-A			-9.286	Likely Pathogenic	0.686	Likely Pathogenic	Likely Benign	0.447	Likely Benign	3.69	Destabilizing	2.9	0.79	Ambiguous	2.24	Destabilizing	0.54	Ambiguous	-0.83	Neutral	0.860	Possibly Damaging	0.354	Benign	3.93	Benign	0.01	Affected	4.32	3	-2	0	-3.1	72.06
c.1202G>A	R401Q (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33438107-G-A			-11.213	Likely Pathogenic	0.969	Likely Pathogenic	Likely Pathogenic	0.780	Likely Pathogenic	0.96	Ambiguous	0.1	1.50	Ambiguous	1.23	Ambiguous	1.20	Destabilizing	-3.69	Deleterious	0.999	Probably Damaging	0.978	Probably Damaging	5.47	Benign	0.04	Affected	3.38	27	1	1	1.0	-28.06
c.1214G>C	R405P (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-14.206	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.572	Likely Pathogenic	3.11	Destabilizing	0.3	5.19	Destabilizing	4.15	Destabilizing	1.26	Destabilizing	-6.32	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.62	Benign	0.01	Affected	3.38	28	-2	0	2.9	-59.07
c.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.1221G>T	Q407H (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-10.526	Likely Pathogenic	0.830	Likely Pathogenic	Ambiguous	0.206	Likely Benign	0.59	Ambiguous	0.0	0.61	Ambiguous	0.60	Ambiguous	1.10	Destabilizing	-4.51	Deleterious	0.982	Probably Damaging	0.947	Probably Damaging	3.88	Benign	0.01	Affected	3.38	28	0	3	0.3	9.01
c.1222A>G	T408A		C2	Uncertain		1				-8.304	Likely Pathogenic	0.114	Likely Benign	Likely Benign	0.118	Likely Benign	0.37	Likely Benign	0.6	-0.06	Likely Benign	0.16	Likely Benign	0.72	Ambiguous	-3.07	Deleterious	0.540	Possibly Damaging	0.131	Benign	4.16	Benign	0.14	Tolerated			1	0	2.5	-30.03
c.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.1240A>G	M414V		GAP	Uncertain		1				-8.003	Likely Pathogenic	0.541	Ambiguous	Likely Benign	0.261	Likely Benign	1.81	Ambiguous	0.4	1.73	Ambiguous	1.77	Ambiguous	0.95	Ambiguous	-2.95	Deleterious	0.999	Probably Damaging	0.987	Probably Damaging	3.43	Benign	0.24	Tolerated			2	1	2.3	-32.06
c.1121C>A	S374Y (3D Viewer)		C2	Uncertain		1				-7.774	In-Between	0.344	Ambiguous	Likely Benign	0.310	Likely Benign	0.71	Ambiguous	1.2	0.66	Ambiguous	0.69	Ambiguous	-0.02	Likely Benign	-1.18	Neutral	0.875	Possibly Damaging	0.271	Benign	5.41	Benign	0.01	Affected	4.32	13	-3	-2	-0.5	76.10	237.3	-76.9	0.5	0.4	0.5	0.3								Uncertain	Ser374 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus, large and relatively hydrophobic residues like tyrosine are rarely tolerated. Additionally, the hydroxyl group of Tyr374 frequently forms various hydrogen bonds with other loop residues in the variant simulations. Although no negative structural effects are observed in the variant simulations, Tyr374 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1136C>G	S379W (3D Viewer)		C2	Uncertain		1	6-33438041-C-G			-8.898	Likely Pathogenic	0.388	Ambiguous	Likely Benign	0.520	Likely Pathogenic	4.32	Destabilizing	3.4	3.56	Destabilizing	3.94	Destabilizing	0.16	Likely Benign	-1.02	Neutral	0.998	Probably Damaging	0.844	Possibly Damaging	3.82	Benign	0.01	Affected	4.32	11	-2	-3	-0.1	99.14	271.3	-75.7	1.4	1.0	0.6	0.5								Uncertain	Ser379 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and thus hydrophobic residues like tryptophan are rarely tolerated. Although no major negative structural effects are observed in the variant simulations, Trp379 may exert drastic effects on the SynGAP-membrane complex dynamics and stability. However, since the effect on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn
c.1339G>C	V447L (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.136	Likely Benign	0.491	Ambiguous	Likely Benign	0.180	Likely Benign	-1.13	Ambiguous	0.1	0.54	Ambiguous	-0.30	Likely Benign	0.03	Likely Benign	-0.29	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.61	Benign	0.90	Tolerated	3.37	32	1	2	-0.4	14.03
c.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.1354G>A	V452I (3D Viewer)		GAP	Uncertain		1				-8.985	Likely Pathogenic	0.361	Ambiguous	Likely Benign	0.218	Likely Benign	-0.08	Likely Benign	0.1	0.51	Ambiguous	0.22	Likely Benign	0.25	Likely Benign	-0.99	Neutral	0.947	Possibly Damaging	0.851	Possibly Damaging	3.26	Benign	0.05	Affected			4	3	0.3	14.03
c.1367A>C	Q456P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.250	Likely Pathogenic	0.993	Likely Pathogenic	Likely Pathogenic	0.469	Likely Benign	3.68	Destabilizing	0.2	8.43	Destabilizing	6.06	Destabilizing	0.82	Ambiguous	-5.66	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	3.34	Benign	0.07	Tolerated	3.37	34	-1	0	1.9	-31.01
c.136C>T	P46S	Likely Benign		Uncertain		1				-3.338	Likely Benign	0.302	Likely Benign	Likely Benign	0.066	Likely Benign										-0.60	Neutral	0.909	Possibly Damaging	0.901	Possibly Damaging	4.15	Benign	0.00	Affected			1	-1	0.8	-10.04
c.1370G>A	S457N	Likely Pathogenic	GAP	Uncertain		1				-10.221	Likely Pathogenic	0.949	Likely Pathogenic	Ambiguous	0.241	Likely Benign	0.19	Likely Benign	0.0	-0.22	Likely Benign	-0.02	Likely Benign	0.67	Ambiguous	-2.76	Deleterious	0.940	Possibly Damaging	0.843	Possibly Damaging	3.28	Benign	0.06	Tolerated			1	1	-2.7	27.03
c.13C>G	R5G	Likely Benign		Uncertain		1				-3.639	Likely Benign	0.150	Likely Benign	Likely Benign	0.169	Likely Benign										-0.16	Neutral	0.013	Benign	0.003	Benign	4.12	Benign	0.00	Affected	4.32	1	-2	-3	4.1	-99.14
c.1153T>C	S385P (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438058-T-C			-5.431	Likely Benign	0.123	Likely Benign	Likely Benign	0.385	Likely Benign	0.91	Ambiguous	0.6	-0.90	Ambiguous	0.01	Likely Benign	0.19	Likely Benign	-0.26	Neutral	0.676	Possibly Damaging	0.693	Possibly Damaging	4.63	Benign	0.04	Affected	4.32	3	1	-1	-0.8	10.04	210.3	18.5	1.8	0.9	0.3	0.0								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycine residues, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Pro385 is potentially tolerated in the Ω loop. However, since the effects on Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.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.1402A>G	M468V (3D Viewer)		GAP	Uncertain		1				-9.461	Likely Pathogenic	0.361	Ambiguous	Likely Benign	0.570	Likely Pathogenic	2.69	Destabilizing	0.1	2.20	Destabilizing	2.45	Destabilizing	0.89	Ambiguous	-1.66	Neutral	0.998	Probably Damaging	0.993	Probably Damaging	-1.21	Pathogenic	0.08	Tolerated	3.37	31	1	2	2.3	-32.06
c.1405G>A	A469T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.540	Likely Pathogenic	0.723	Likely Pathogenic	Likely Benign	0.527	Likely Pathogenic	2.26	Destabilizing	0.1	1.90	Ambiguous	2.08	Destabilizing	0.34	Likely Benign	-1.46	Neutral	0.994	Probably Damaging	0.986	Probably Damaging	-1.21	Pathogenic	0.42	Tolerated			1	0	-2.5	30.03
c.1408A>G	M470V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.856	Likely Pathogenic	0.478	Ambiguous	Likely Benign	0.770	Likely Pathogenic	2.73	Destabilizing	0.1	1.88	Ambiguous	2.31	Destabilizing	1.31	Destabilizing	-3.58	Deleterious	0.999	Probably Damaging	0.993	Probably Damaging	-1.20	Pathogenic	0.15	Tolerated	3.37	34	1	2	2.3	-32.06
c.1169G>A	G390E (3D Viewer)		C2	Uncertain		1				-7.913	In-Between	0.646	Likely Pathogenic	Likely Benign	0.575	Likely Pathogenic	2.61	Destabilizing	0.9	4.28	Destabilizing	3.45	Destabilizing	0.47	Likely Benign	-0.87	Neutral	0.276	Benign	0.045	Benign	1.32	Pathogenic	0.05	Affected	4.32	8	0	-2	-3.1	72.06	241.5	-108.4	0.6	0.5	-0.1	0.1								Uncertain	Gly390 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364 and res. Ala399-Ile411). The Ω loop is assumed to directly interact with the membrane, and it is observed to move arbitrarily throughout the WT solvent simulations. This loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play significant roles in protein functions that require flexibility, and so they are rich in glycine residues, prolines, and to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Glu390 may not be as well tolerated in the Ω loop. Additionally, the carboxylate group of Glu390 occasionally forms H-bonds with other loop residues in the variant simulations. The interaction between the acidic carboxylate side chain and the acidic membrane lipids may further influence the SynGAP-membrane complex. However, since the effects on the Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1436G>C	R479P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.795	Likely Pathogenic	0.938	Likely Pathogenic	Ambiguous	0.277	Likely Benign	2.86	Destabilizing	0.2	3.88	Destabilizing	3.37	Destabilizing	0.81	Ambiguous	-3.52	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.41	Benign	0.18	Tolerated			0	-2	2.9	-59.07
c.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.1453C>A	R485S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.603	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.609	Likely Pathogenic	0.40	Likely Benign	0.1	1.07	Ambiguous	0.74	Ambiguous	0.82	Ambiguous	-5.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	1.93	Pathogenic	0.00	Affected			0	-1	3.7	-69.11
c.1468G>C	A490P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.905	Likely Pathogenic	0.941	Likely Pathogenic	Ambiguous	0.878	Likely Pathogenic	-1.27	Ambiguous	0.1	1.31	Ambiguous	0.02	Likely Benign	1.07	Destabilizing	-4.81	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.42	Pathogenic	0.01	Affected	3.37	35	-1	1	-3.4	26.04
c.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.1199T>A	V400E (3D Viewer)	Likely Pathogenic	C2	Uncertain		1				-13.686	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.810	Likely Pathogenic	3.70	Destabilizing	0.2	2.46	Destabilizing	3.08	Destabilizing	2.29	Destabilizing	-4.88	Deleterious	0.920	Possibly Damaging	0.335	Benign	5.31	Benign	0.00	Affected	3.38	27	-2	-2	-7.7	29.98	249.1	-38.8	-0.1	0.1	1.0	0.0	X		X				X	Potentially Pathogenic	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). In the variant simulations, the negatively charged carboxylate group of the Glu400 side chain is not suitable for occupying the hydrophobic niche. Consequently, the side chain escapes the center of the C2 domain and interacts with the backbone amide groups of Leu402 in the same β strand and/or Ile269 and Glu270 in a neighboring β strand (res. Arg259-Arg272). This residue swap disrupts the hydrophobic packing and generally has extensive negative effects on the C2 domain structure. At a minimum, the residue swap could affect the C2 domain stability and membrane association.
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.1474A>G	K492E (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-16.175	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.510	Likely Pathogenic	1.53	Ambiguous	0.1	1.90	Ambiguous	1.72	Ambiguous	1.42	Destabilizing	-3.98	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.99	Benign	0.01	Affected	3.37	35	1	0	0.4	0.94
c.1483G>A	E495K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.478	Likely Pathogenic	0.986	Likely Pathogenic	Likely Pathogenic	0.869	Likely Pathogenic	0.15	Likely Benign	0.2	0.66	Ambiguous	0.41	Likely Benign	0.70	Ambiguous	-3.91	Deleterious	0.999	Probably Damaging	0.994	Probably Damaging	-1.29	Pathogenic	0.01	Affected	3.37	35	1	0	-0.4	-0.94
c.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.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.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.1256A>G	E419G (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.589	Likely Pathogenic	0.956	Likely Pathogenic	Likely Pathogenic	0.469	Likely Benign	1.41	Ambiguous	0.0	1.94	Ambiguous	1.68	Ambiguous	0.83	Ambiguous	-6.42	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	3.31	Benign	0.02	Affected	3.37	29	0	-2	3.1	-72.06	165.3	110.8	0.0	0.0	-0.1	0.0		X						Potentially Pathogenic	The carboxylate group of Glu419, located on an α helix (res. Met414-Glu436), forms a salt bridge with the side chain of either Arg716 or Lys418 from an opposing helix (res. Pro713-Arg726). The backbone amide group of Glu419 does not form H-bonds, resulting in a slight bend in the α helix. Thus, although glycine is known as an “α helix breaker,” the residue swap does not disrupt the continuity or integrity of the α helix. However, because Gly419 cannot form a salt bridge with the guanidinium group of the Arg716 side chain, the C2-GAP domain tertiary structure could be compromised during folding.
c.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.1260T>G	F420L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.432	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.146	Likely Benign	1.76	Ambiguous	0.0	1.41	Ambiguous	1.59	Ambiguous	1.04	Destabilizing	-5.39	Deleterious	0.009	Benign	0.005	Benign	4.22	Benign	0.39	Tolerated	3.37	29	2	0	1.0	-34.02	231.1	13.2	0.0	0.0	-0.1	0.0						X		Potentially Benign	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). In the variant simulations, the iso-butyl side chain of Leu420 also packs into the hydrophobic inter-helix niche, but due to its smaller size, the resulting steric interactions are not as favorable as with phenylalanine. In short, the residue swap does not cause severe effects on the protein structure based on 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.1516C>T	L506F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.262	Likely Pathogenic	0.883	Likely Pathogenic	Ambiguous	0.464	Likely Benign	4.92	Destabilizing	0.8	5.76	Destabilizing	5.34	Destabilizing	0.91	Ambiguous	-3.98	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.62	Pathogenic	0.01	Affected	3.37	35	0	2	-1.0	34.02
c.1540A>T	I514F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.383	Likely Pathogenic	0.962	Likely Pathogenic	Likely Pathogenic	0.601	Likely Pathogenic	2.35	Destabilizing	0.3	3.74	Destabilizing	3.05	Destabilizing	0.93	Ambiguous	-3.98	Deleterious	0.997	Probably Damaging	0.993	Probably Damaging	2.89	Benign	0.00	Affected	3.37	35	0	1	-1.7	34.02
c.1552T>C	Y518H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.797	Likely Pathogenic	0.943	Likely Pathogenic	Ambiguous	0.496	Likely Benign	2.39	Destabilizing	0.4	0.82	Ambiguous	1.61	Ambiguous	1.31	Destabilizing	-4.74	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.40	Benign	0.08	Tolerated			0	2	-1.9	-26.03
c.1558T>C	S520P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.707	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.855	Likely Pathogenic	3.72	Destabilizing	0.8	8.86	Destabilizing	6.29	Destabilizing	0.83	Ambiguous	-4.57	Deleterious	0.997	Probably Damaging	0.986	Probably Damaging	-1.32	Pathogenic	0.01	Affected			1	-1	-0.8	10.04
c.1559C>T	S520F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.541	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.833	Likely Pathogenic	-1.20	Ambiguous	0.4	0.39	Likely Benign	-0.41	Likely Benign	0.25	Likely Benign	-5.57	Deleterious	0.999	Probably Damaging	0.996	Probably Damaging	-1.36	Pathogenic	0.00	Affected	3.37	35	-2	-3	3.6	60.10
c.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.1304T>G	L435W (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.889	Likely Pathogenic	0.992	Likely Pathogenic	Likely Pathogenic	0.572	Likely Pathogenic	2.11	Destabilizing	0.1	0.69	Ambiguous	1.40	Ambiguous	1.66	Destabilizing	-5.63	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.15	Benign	0.00	Affected	3.37	29	-2	-2	-4.7	73.05	242.2	-25.2	0.0	0.0	0.3	0.1							X	Potentially Pathogenic	The iso-butyl side chain of Leu435, located in an α helix (res. Met414-Glu436), packs against other hydrophobic residues in an interhelix space (e.g., Val699, Val447, Leu489, Leu439) in the WT simulations. In the variant simulations, the indole ring of Trp435 fits into the same niche despite its considerably bulkier size. Additionally, the side chain forms an H-bond with the backbone carbonyl of Leu696 in an α helix (res. Asp684-Gln702). Although no apparent negative changes are observed during the variant simulation, the size difference between the swapped residues could affect the protein folding process.
c.1306G>A	E436K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.869	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.829	Likely Pathogenic	0.56	Ambiguous	0.1	2.86	Destabilizing	1.71	Ambiguous	0.82	Ambiguous	-3.77	Deleterious	0.994	Probably Damaging	0.951	Probably Damaging	4.71	Benign	0.02	Affected	3.37	29	0	1	-0.4	-0.94	186.8	39.8	0.0	0.0	-0.2	0.0	X	X		X				Potentially Pathogenic	The carboxylate group of Glu436, located on the α helix (res. Met414-Glu436), forms a salt bridge with the amino group of the Lys444 side chain on an opposing α helix (res. Val441-Ser457). The backbone carbonyl of Glu436 also H-bonds with the Lys444 side chain, which helps keep the ends of the two α helices tightly connected. In contrast, in the variant simulations, the salt bridge formation with Lys444 is not possible. Instead, the repelled Lys436 side chain rotates outward, causing a change in the α helix backbone H-bonding: the amide group of Lys444 H-bonds with the carbonyl of Ala433 instead of the carbonyl of Cys432.
c.1606T>G	L536V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.014	Likely Pathogenic	0.269	Likely Benign	Likely Benign	0.586	Likely Pathogenic	1.25	Ambiguous	0.3	1.22	Ambiguous	1.24	Ambiguous	1.20	Destabilizing	-2.81	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	-1.34	Pathogenic	0.09	Tolerated	3.37	34	2	1	0.4	-14.03	204.7	26.4	0.2	0.0	-0.2	0.2						X		Potentially Benign	Leu536 is located on an α-helix (res. Ala533-Val560) at the membrane interface. The iso-butyl group of Leu536 interacts with nearby hydrophobic residues in the preceding loop (e.g., Val526, Pro528, Cys531). In the variant simulations, the iso-propyl side chain of Val536 forms similar hydrophobic interactions as Leu536 in the WT, causing no negative structural effects.
c.1349C>A	A450E (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.578	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.653	Likely Pathogenic	3.86	Destabilizing	0.2	5.23	Destabilizing	4.55	Destabilizing	1.59	Destabilizing	-4.67	Deleterious	0.999	Probably Damaging	0.992	Probably Damaging	3.38	Benign	0.07	Tolerated	3.37	32	0	-1	-5.3	58.04	240.1	-82.6	0.0	0.0	0.7	0.0			X				X	Potentially Pathogenic	The methyl group of Ala450, located in an α helix (res. Asn440-Thr458), packs against hydrophobic residues in the inter-helix space (e.g., Leu692). In the variant simulations, the carboxylate group of the Glu450 side chain rotates outward, away from the hydrophobic niche, where it does not form any lasting salt bridges or H-bonds. Although the residue swap does not negatively affect the protein structure based on the simulations, it is possible that the introduction of the negatively charged residue adversely affects the folding process or tertiary assembly.
c.1354G>T	V452F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.769	Likely Pathogenic	0.975	Likely Pathogenic	Likely Pathogenic	0.511	Likely Pathogenic	9.21	Destabilizing	0.1	0.37	Likely Benign	4.79	Destabilizing	0.61	Ambiguous	-4.94	Deleterious	0.999	Probably Damaging	0.993	Probably Damaging	3.29	Benign	0.00	Affected	3.37	34	-1	-1	-1.4	48.04	249.4	-35.7	0.0	0.0	0.4	0.1							X	Potentially Pathogenic	The iso-propyl side chain of Val452, located in the middle of an α helix (res. Val441-Ser457), packs against hydrophobic residues in the inter-helix space at the intersection of three α helices (e.g., Leu500, His453, Leu465). In the variant simulations, the larger side chain of Phe452 cannot pack against the opposing α helix (res. Leu489-Glu519) as efficiently as valine. Due to space restrictions, the phenol ring adjusts to make room by rotating slightly sideways in the inter-helix space. Besides this small and local shift, no large-scale effects on the protein structure are seen based on the simulations. However, the size difference between the swapped residues could affect the protein folding process.
c.1390T>G	F464V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.254	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.592	Likely Pathogenic	3.61	Destabilizing	0.1	2.89	Destabilizing	3.25	Destabilizing	1.40	Destabilizing	-6.96	Deleterious	0.998	Probably Damaging	0.996	Probably Damaging	3.36	Benign	0.04	Affected	3.37	34	-1	-1	1.4	-48.04	210.1	40.5	-0.1	0.0	-0.9	0.3							X	Potentially Pathogenic	The phenyl ring of Phe464, located in the middle of an α helix (res. Ala461–Phe476), packs against hydrophobic residues (e.g., Met468, Leu451, Leu455, and Tyr428) in the inter-helix space formed with two other α helices (res. Asn440-Lys460 and res. Pro413-Glu436). The iso-propyl side chain of Val464 is similarly hydrophobic but considerably smaller than the original phenyl ring of Phe464. To compensate for the size difference, neighboring residues need to fill in the gap in the variant simulations.The phenolic side chain of Tyr428, located at the middle bend of an α helix (res. Glu436-Pro413), assumes a new position in the inter-helix space or rotates inward next to the third α helix (res. Asn440-Lys460) when the stable H-bond between Tyr428 and Asp467 seen in the WT simulations breaks. The residue swap also leads to the loss of the methionine-aromatic interaction between the Met468 and Phe464 side chains, which could weaken the integrity of the parent α helix (res. Ala461-Phe476). Although the simulations likely underestimate the full adverse effect of the introduced mutation during folding, the two opposing α helices (res. Ala461–Phe476 and res. Glu436-Pro413) move substantially closer to each other in the variant simulations.
c.1393C>G	L465V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.893	Likely Pathogenic	0.838	Likely Pathogenic	Ambiguous	0.276	Likely Benign	2.46	Destabilizing	0.1	2.66	Destabilizing	2.56	Destabilizing	1.21	Destabilizing	-2.98	Deleterious	0.996	Probably Damaging	0.992	Probably Damaging	2.44	Pathogenic	0.10	Tolerated	3.37	34	2	1	0.4	-14.03	204.3	30.9	0.0	0.0	-0.4	0.6						X		Potentially Benign	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 iso-propyl side chain of Val465 is equally sized and similarly hydrophobic as the original side chain of Leu465. Hence, the mutation does not exert any negative effects on the protein structure based on the variant simulations.
c.1635G>A	M545I (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.348	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.592	Likely Pathogenic	0.47	Likely Benign	0.1	0.14	Likely Benign	0.31	Likely Benign	0.63	Ambiguous	-3.61	Deleterious	0.935	Possibly Damaging	0.941	Probably Damaging	-1.27	Pathogenic	0.28	Tolerated	3.37	35	1	2	2.6	-18.03
c.1663G>A	V555I (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.544	Likely Benign	0.084	Likely Benign	Likely Benign	0.253	Likely Benign	-0.82	Ambiguous	0.0	-0.41	Likely Benign	-0.62	Ambiguous	-0.55	Ambiguous	0.45	Neutral	0.002	Benign	0.002	Benign	-1.26	Pathogenic	1.00	Tolerated			4	3	0.3	14.03
c.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.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.1406C>A	A469D (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.643	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.738	Likely Pathogenic	5.09	Destabilizing	0.2	4.16	Destabilizing	4.63	Destabilizing	1.68	Destabilizing	-3.48	Deleterious	0.999	Probably Damaging	0.996	Probably Damaging	-1.34	Pathogenic	0.21	Tolerated	3.37	34	0	-2	-5.3	44.01	237.0	-58.2	-0.2	0.1	0.8	0.1	X		X					Potentially Pathogenic	The methyl group of Ala469, located in an α helix (res. Ala461–Phe476), interacts with hydrophobic residues (e.g., Trp572, Leu588, Met470) in an inter-helix space formed by two other α helices (res. Glu582–Ser604, res. Arg563–Gly580). In the variant simulations, Asp469 introduces a negatively charged and bulky side chain into the hydrophobic niche. Consequently, the side chain of Asp469 rotates outward, allowing the carboxylate group to form a salt bridge with the guanidinium group of Arg575 on the protein surface. This interaction affects the continuity of the parent α helix (Ala461–Phe476). Due to the importance of hydrophobic packing, the structural effects could be more pronounced during actual protein folding.
c.1673A>G	H558R (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.445	Likely Pathogenic	0.554	Ambiguous	Likely Benign	0.587	Likely Pathogenic	-1.14	Ambiguous	0.1	-0.23	Likely Benign	-0.69	Ambiguous	1.03	Destabilizing	-4.94	Deleterious	0.677	Possibly Damaging	0.239	Benign	-1.24	Pathogenic	0.14	Tolerated	3.37	35	0	2	-1.3	19.05
c.169C>T	L57F	Likely Benign		Uncertain		2				-5.096	Likely Benign	0.459	Ambiguous	Likely Benign	0.051	Likely Benign										-0.78	Neutral	0.824	Possibly Damaging	0.879	Possibly Damaging	3.96	Benign	0.00	Affected	4.32	1	2	0	-1.0	34.02
c.1702G>T	V568L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.503	Likely Pathogenic	0.921	Likely Pathogenic	Ambiguous	0.651	Likely Pathogenic	-0.30	Likely Benign	0.3	0.57	Ambiguous	0.14	Likely Benign	0.56	Ambiguous	-2.69	Deleterious	0.511	Possibly Damaging	0.147	Benign	-1.23	Pathogenic	0.04	Affected	3.37	35	1	2	-0.4	14.03
c.1409T>C	M470T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.104	Likely Pathogenic	0.976	Likely Pathogenic	Likely Pathogenic	0.763	Likely Pathogenic	3.19	Destabilizing	0.1	2.68	Destabilizing	2.94	Destabilizing	1.49	Destabilizing	-5.30	Deleterious	0.996	Probably Damaging	0.985	Probably Damaging	-1.08	Pathogenic	0.24	Tolerated	3.37	34	-1	-1	-2.6	-30.09	213.8	46.5	0.0	0.0	-0.2	0.2		X					X	Potentially Pathogenic	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, Cys576, Trp572) formed by two other α helices (res. Ser604–Arg581, res. Pro562–Arg579). In the WT simulations, the Met470 side chain 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 hydroxyl group of the Thr470 side chain forms an H-bond with the backbone carbonyl group of Ser466 in the α helix, potentially lowering its structural integrity. Importantly, the hydroxyl group of Thr470 also forms an H-bond with the guanidinium group of Arg575, which helps it form a more permanent salt bridge with Asp467.
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.172A>G	M58V	Likely Benign		Uncertain		1				-2.211	Likely Benign	0.688	Likely Pathogenic	Likely Benign	0.160	Likely Benign										-0.71	Neutral	0.006	Benign	0.091	Benign	4.19	Benign	0.00	Affected	4.32	1	1	2	2.3	-32.06
c.1763T>C	L588P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.771	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.932	Likely Pathogenic	5.61	Destabilizing	0.5	12.91	Destabilizing	9.26	Destabilizing	2.33	Destabilizing	-6.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.42	Pathogenic	0.00	Affected	3.38	34	-3	-3	-5.4	-16.04
c.1778T>C	L593P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.961	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.777	Likely Pathogenic	5.75	Destabilizing	0.9	10.77	Destabilizing	8.26	Destabilizing	2.43	Destabilizing	-6.77	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.77	Benign	0.00	Affected			-3	-3	-5.4	-16.04
c.1456G>A	E486K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.545	Likely Pathogenic	0.988	Likely Pathogenic	Likely Pathogenic	0.435	Likely Benign	0.06	Likely Benign	0.1	0.37	Likely Benign	0.22	Likely Benign	0.41	Likely Benign	-3.58	Deleterious	1.000	Probably Damaging	0.988	Probably Damaging	3.40	Benign	0.12	Tolerated	3.37	35	0	1	-0.4	-0.94	206.8	52.1	-0.3	0.1	0.2	0.0	X			X				Uncertain	Glu486 is located in an α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. It is adjacent to the arginine finger (Arg485) and is expected to closely interact with Ras. The residue swap could affect complex formation with the GTPase and its activation. In the WT simulations, the carboxylate group of Glu486 forms salt bridges with Arg485 and Arg475 on the preceding α-helix (res. Ala461-Phe476). In the variant simulations, Lys486 does not form any specific interactions. Although the amino group of the Lys486 side chain cannot form these salt bridges, no negative effects on the protein structure are observed. Nevertheless, the potential role of Glu486 in SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations, and no definite conclusions can be drawn.
c.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.
c.1784T>A	L595Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.101	Likely Pathogenic	0.984	Likely Pathogenic	Likely Pathogenic	0.733	Likely Pathogenic	0.79	Ambiguous	0.1	1.40	Ambiguous	1.10	Ambiguous	1.99	Destabilizing	-5.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.75	Benign	0.00	Affected	3.37	35	-2	-2	-7.3	14.97
c.1784T>C	L595P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.856	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.747	Likely Pathogenic	2.09	Destabilizing	0.8	5.88	Destabilizing	3.99	Destabilizing	1.78	Destabilizing	-6.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.72	Benign	0.00	Affected	3.37	35	-3	-3	-5.4	-16.04
c.1789T>C	F597L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.173	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.929	Likely Pathogenic	0.74	Ambiguous	0.1	2.12	Destabilizing	1.43	Ambiguous	1.20	Destabilizing	-5.97	Deleterious	0.999	Probably Damaging	0.994	Probably Damaging	-2.06	Pathogenic	0.13	Tolerated			2	0	1.0	-34.02
c.1792C>G	L598V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.002	Likely Pathogenic	0.578	Likely Pathogenic	Likely Benign	0.221	Likely Benign	1.89	Ambiguous	0.1	1.58	Ambiguous	1.74	Ambiguous	1.01	Destabilizing	-2.92	Deleterious	0.944	Possibly Damaging	0.786	Possibly Damaging	3.21	Benign	0.02	Affected	3.37	35	2	1	0.4	-14.03	218.4	29.6	0.0	0.0	0.8	0.0						X		Potentially Benign	The iso-butyl side chain of Leu598, located on an α helix (res. Glu582-Met603), packs hydrophobically with other hydrophobic residues in the inter-helix space (e.g., Ile602, Phe594, Ile510).In the variant simulations, Val598, which has similar size and physicochemical properties to leucine, resides in the inter-helix hydrophobic space in a similar manner to Leu598 in the WT. This causes no negative effects on the protein structure.
c.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.1485A>C	E495D (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-3.574	Likely Benign	0.958	Likely Pathogenic	Likely Pathogenic	0.566	Likely Pathogenic	1.39	Ambiguous	0.1	1.03	Ambiguous	1.21	Ambiguous	0.98	Ambiguous	-2.52	Deleterious	0.998	Probably Damaging	0.989	Probably Damaging	-1.41	Pathogenic	0.17	Tolerated	3.37	35	3	2	0.0	-14.03	220.6	38.8	0.0	0.0	0.1	0.1		X		X				Uncertain	Glu495 is located in the α-helix (res. Leu489-Glu519), and its carboxylate group forms salt bridges with the neighboring Lys492 and with Arg596 on an opposing α-helix (res. Glu582-Met603) in the WT simulations. In the variant simulations, the acidic carboxylate side chain of Asp495 can also form salt bridges with both Lys492 and Arg596. However, the shorter side chain of aspartate tends to favor forming a salt bridge with the nearby Arg499 on the same α-helix instead. Asp495 might not maintain the salt bridge with Arg596 on the opposing α-helix as efficiently as Glu495 in the WT, potentially weakening the tertiary structure. Regardless, the potential negative effect is likely to be minor, with no deleterious effects observed on the protein structure during the simulations. However, due to its location at the GAP-Ras interface, the effect of the residue swap on SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.1487A>G	E496G (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.529	Likely Pathogenic	0.850	Likely Pathogenic	Ambiguous	0.825	Likely Pathogenic	1.83	Ambiguous	0.1	1.76	Ambiguous	1.80	Ambiguous	0.92	Ambiguous	-6.16	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.45	Pathogenic	0.02	Affected	3.37	35	0	-2	3.1	-72.06	173.9	103.1	0.0	0.0	-0.7	0.0		X		X				Potentially Pathogenic	Glu496 is located in the α-helix (res. Leu489-Glu519), and its carboxylate group forms salt bridges with the neighbouring residues Lys492 and Arg499 in the WT simulations. Glu496 also forms a hydrogen bond with Ser449 on an opposing helix (res. Val441-Ser457). In the variant simulations, Gly496 cannot form these salt bridges, which could weaken the secondary structure. Additionally, the loss of the hydrogen bond with Ser449 on the opposite helix can weaken the tertiary structure assembly. Moreover, glycine is an α-helix breaker, and it is seen to weaken the integrity of the helix as the hydrogen bonding between the backbone atoms of Gly496 and Ala493 breaks down. Also, due to its location at the GAP-Ras interface, the interaction of Glu496 with Arg499 and Lys492 might play a role in complex association and stability, which cannot be fully addressed using the SynGAP solvent-only simulations.
c.1490A>G	Y497C (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.872	Likely Pathogenic	0.948	Likely Pathogenic	Ambiguous	0.806	Likely Pathogenic	3.88	Destabilizing	0.1	4.76	Destabilizing	4.32	Destabilizing	1.40	Destabilizing	-8.82	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.65	Pathogenic	0.03	Affected	3.37	35	0	-2	3.8	-60.04	209.9	59.1	-0.1	0.0	-0.3	0.1		X					X	Potentially Pathogenic	Tyr497 is located in the α-helix (res. Leu489-Glu519) within the inter-helix space of four α-helices (res. Leu489-Ile501, res. Val441-Ser457, res. Arg563-Glu578, res. Ala461-Val473). In the WT simulations, the phenol ring of Tyr497 hydrophobically packs with other residues in the inter-helix space (e.g., Leu465, Leu565, Val568). The hydroxyl group of Tyr497 also alternately forms hydrogen bonds with the carboxylate side chain of Gln456 and the backbone carbonyl of Glu564. Thus, Tyr497 plays a role in the folding and maintenance of the tertiary structure assembly between these four helices.In the variant simulations, the comparatively smaller residue, Cys497, cannot maintain any of the interactions seen with Tyr497 in the WT. Although no severe deleterious consequences are observed in the simulations, the structural effects could be more pronounced during actual protein folding. Indeed, the tertiary structure is seen to slightly break apart in the variant simulations.
c.182A>C	E61A	Likely Benign		Uncertain		1				-5.235	Likely Benign	0.453	Ambiguous	Likely Benign	0.074	Likely Benign										-1.52	Neutral	0.458	Possibly Damaging	0.678	Possibly Damaging	4.12	Benign	0.00	Affected			0	-1	5.3	-58.04
c.1835A>C	Q612P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.684	Likely Pathogenic	0.673	Likely Pathogenic	Likely Benign	0.671	Likely Pathogenic	-0.19	Likely Benign	0.3	3.06	Destabilizing	1.44	Ambiguous	0.56	Ambiguous	-5.84	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.31	Pathogenic	0.19	Tolerated			0	-1	1.9	-31.01
c.1851G>T	E617D (3D Viewer)	Likely Benign	GAP	Uncertain		1				-1.349	Likely Benign	0.241	Likely Benign	Likely Benign	0.322	Likely Benign	0.12	Likely Benign	0.1	0.80	Ambiguous	0.46	Likely Benign	0.07	Likely Benign	-0.01	Neutral	0.994	Probably Damaging	0.979	Probably Damaging	-1.35	Pathogenic	0.88	Tolerated	3.37	35	2	3	0.0	-14.03
c.1855A>T	T619S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.608	Likely Pathogenic	0.677	Likely Pathogenic	Likely Benign	0.602	Likely Pathogenic	1.09	Ambiguous	0.2	1.35	Ambiguous	1.22	Ambiguous	0.85	Ambiguous	-3.42	Deleterious	0.999	Probably Damaging	0.998	Probably Damaging	-1.30	Pathogenic	0.05	Affected	3.37	35	1	1	-0.1	-14.03
c.1873C>G	L625V	Likely Pathogenic	GAP	Uncertain		1				-11.319	Likely Pathogenic	0.833	Likely Pathogenic	Ambiguous	0.480	Likely Benign	1.80	Ambiguous	0.7	1.69	Ambiguous	1.75	Ambiguous	1.42	Destabilizing	-2.96	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	3.07	Benign	0.01	Affected			2	1	0.4	-14.03
c.1502T>C	I501T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.996	Likely Benign	0.252	Likely Benign	Likely Benign	0.362	Likely Benign	2.40	Destabilizing	0.1	1.81	Ambiguous	2.11	Destabilizing	1.57	Destabilizing	-3.48	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.44	Benign	0.16	Tolerated	3.37	35	0	-1	-5.2	-12.05	214.5	26.9	0.0	0.0	0.5	0.0							X	Potentially Pathogenic	Ile501 is located near a hinge in the middle of an α-helix (res. Leu489-Glu519). The sec-butyl side chain of Ile501 is hydrophobically packed with other residues in the inter-helix space (e.g., Leu500, Tyr497, Phe679) in the WT simulations. In the variant simulations, the hydroxyl group of Thr501 forms a hydrogen bond with the backbone atoms of Tyr497 on the same α-helix, which may weaken the α-helix integrity. Additionally, the polar hydroxyl group of Thr501 is not suitable for the hydrophobic inter-helix space, and thus, the residue swap could affect protein folding. However, Ile501 is followed by Gly502, which facilitates a hinge in the middle of the α-helix, making further weakening caused by Thr501 unlikely to be harmful to the α-helix integrity.
c.1505G>A	G502D (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.796	Likely Pathogenic	0.994	Likely Pathogenic	Likely Pathogenic	0.915	Likely Pathogenic	3.79	Destabilizing	0.9	5.69	Destabilizing	4.74	Destabilizing	1.38	Destabilizing	-6.80	Deleterious	0.999	Probably Damaging	0.977	Probably Damaging	-1.66	Pathogenic	0.00	Affected	3.37	35	1	-1	-3.1	58.04	224.2	-80.0	-0.8	0.7	0.6	0.3	X		X				X	Potentially Pathogenic	Gly502 is located in a hinge in the middle of an α-helix (res. Leu489-Glu519). In the WT, Gly502 acts as an α-helix breaker due to its lack of a side chain, facilitating a bend in the middle of the α-helix. In the variant simulations, the carboxylate group of Asp502 forms hydrogen bonds with neighboring residues (e.g., Ser677, Lys504), disrupting the hinge. Additionally, Asp502 struggles to fit into the α-helix hinge and cannot generate a similar bend as Gly502, which would drastically affect the secondary structure during folding. Thus, the deleterious effect seen in the simulations is likely an underestimate of the impact of the residue swap on the protein structure 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.1877T>C	I626T	Likely Pathogenic	GAP	Uncertain		1				-10.420	Likely Pathogenic	0.946	Likely Pathogenic	Ambiguous	0.640	Likely Pathogenic	2.94	Destabilizing	0.1	2.70	Destabilizing	2.82	Destabilizing	2.23	Destabilizing	-4.18	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	3.04	Benign	0.00	Affected			0	-1	-5.2	-12.05
c.187G>A	E63K	Likely Benign		Uncertain		1				-4.976	Likely Benign	0.894	Likely Pathogenic	Ambiguous	0.103	Likely Benign										-0.70	Neutral	0.458	Possibly Damaging	0.678	Possibly Damaging	3.98	Benign	0.00	Affected	4.32	1	1	0	-0.4	-0.94
c.187G>C	E63Q	Likely Benign		Uncertain		1				-4.038	Likely Benign	0.687	Likely Pathogenic	Likely Benign	0.078	Likely Benign										-0.85	Neutral	0.659	Possibly Damaging	0.775	Possibly Damaging	3.90	Benign	0.00	Affected	4.32	1	2	2	0.0	-0.98
c.1913A>G	K638R (3D Viewer)	Likely Benign	GAP	Uncertain		1				-2.700	Likely Benign	0.110	Likely Benign	Likely Benign	0.216	Likely Benign	0.09	Likely Benign	0.1	-0.04	Likely Benign	0.03	Likely Benign	0.53	Ambiguous	-2.55	Deleterious	0.649	Possibly Damaging	0.240	Benign	3.41	Benign	0.13	Tolerated	3.37	31	2	3	-0.6	28.01
c.1942T>C	F648L	Likely Pathogenic	GAP	Uncertain		1				-9.296	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.468	Likely Benign	2.71	Destabilizing	0.8	2.08	Destabilizing	2.40	Destabilizing	1.04	Destabilizing	-5.98	Deleterious	0.999	Probably Damaging	0.976	Probably Damaging	3.45	Benign	0.08	Tolerated			2	0	1.0	-34.02
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.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.1579G>T	D527Y (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.386	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.905	Likely Pathogenic	-0.77	Ambiguous	0.2	1.89	Ambiguous	0.56	Ambiguous	-0.14	Likely Benign	-8.79	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-2.41	Pathogenic	0.00	Affected	3.37	35	-4	-3	2.2	48.09	270.9	-45.7	0.1	0.1	-0.1	0.0		X						Potentially Pathogenic	Asp527 is located on an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the carboxylate group of the Asp527 side chain forms hydrogen bonds with the backbone atoms of loop residues (e.g., Ile529, Lys530) facing the membrane surface. In the variant simulations, Tyr527 is a bulkier residue that faces away from the loop and stacks with Phe646 in a nearby α-helix (res. Ser614-Ser668). Regardless, no negative structural effects are observed during the variant simulations. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.
c.1586T>C	I529T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-0.539	Likely Benign	0.336	Likely Benign	Likely Benign	0.343	Likely Benign	0.22	Likely Benign	0.2	0.16	Likely Benign	0.19	Likely Benign	0.17	Likely Benign	0.24	Neutral	0.872	Possibly Damaging	0.820	Possibly Damaging	-1.23	Pathogenic	0.55	Tolerated	3.37	35	0	-1	-5.2	-12.05	207.2	29.8	0.2	0.0	0.2	0.1						X		Potentially Benign	Ile529 is located on an α-α loop between the two α-helices (res. Gly502-Tyr518 and Ala533-Val560). In the WT simulations, the sec-butyl side chain of Ile529 faces the membrane interface and shows no specific interactions. In the variant simulations, the hydroxyl group of Thr529 forms a hydrogen bond with the carboxylate side chain of Asp527, but no negative structural changes are observed. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.
c.1957C>G	L653V	Likely Benign	GAP	Uncertain		1				-7.050	In-Between	0.301	Likely Benign	Likely Benign	0.146	Likely Benign	3.28	Destabilizing	0.3	2.18	Destabilizing	2.73	Destabilizing	1.32	Destabilizing	-2.25	Neutral	0.227	Benign	0.039	Benign	3.28	Benign	0.08	Tolerated			2	1	0.4	-14.03
c.1964T>A	L655Q (3D Viewer)	Likely Benign	GAP	Uncertain		1				-5.278	Likely Benign	0.144	Likely Benign	Likely Benign	0.139	Likely Benign	-0.01	Likely Benign	0.0	0.69	Ambiguous	0.34	Likely Benign	-0.15	Likely Benign	0.61	Neutral	0.955	Possibly Damaging	0.602	Possibly Damaging	3.59	Benign	0.65	Tolerated	3.39	24	-2	-2	-7.3	14.97	229.9	-8.6	0.0	0.0	0.4	0.0						X		Potentially Benign	The iso-butyl side chain of Leu655, located on the surface of an α helix (res. Ser641-Glu666), is not involved in any interactions in the WT simulations. In the variant simulations, the carboxamide side chain of Gln655 dynamically interacts with neighboring residues (e.g., Glu651, Glu656, Arg544) on the protein surface, with no negative structural effects.
c.196C>G	P66A	Likely Benign		Uncertain		1				-2.845	Likely Benign	0.891	Likely Pathogenic	Ambiguous	0.091	Likely Benign										-1.56	Neutral	0.805	Possibly Damaging	0.539	Possibly Damaging	4.04	Benign	0.00	Affected	4.32	1	1	-1	3.4	-26.04
c.1970G>T	W657L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.411	Likely Pathogenic	0.960	Likely Pathogenic	Likely Pathogenic	0.213	Likely Benign	0.14	Likely Benign	0.1	0.73	Ambiguous	0.44	Likely Benign	0.87	Ambiguous	-10.86	Deleterious	0.277	Benign	0.078	Benign	3.52	Benign	0.14	Tolerated	3.39	24	-2	-2	4.7	-73.05
c.1971G>C	W657C	Likely Pathogenic	GAP	Uncertain		1				-12.035	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.463	Likely Benign	2.74	Destabilizing	0.3	1.69	Ambiguous	2.22	Destabilizing	1.30	Destabilizing	-11.06	Deleterious	1.000	Probably Damaging	0.982	Probably Damaging	3.43	Benign	0.03	Affected			-8	-2	3.4	-83.07
c.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.1621G>C	A541P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.733	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.594	Likely Pathogenic	2.47	Destabilizing	0.3	7.26	Destabilizing	4.87	Destabilizing	0.86	Ambiguous	-3.16	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.34	Pathogenic	0.07	Tolerated	3.37	35	1	-1	-3.4	26.04	170.4	-11.2	0.1	0.0	0.1	0.0	X							Potentially Pathogenic	Ala541 is located on the outer surface of an α-helix (res. Ala533-Val560). The methyl group of Ala541 is on the surface and does not form any interactions. Proline lacks a free backbone amide group, and thus, Pro541 is unable to form a hydrogen bond with the carbonyl group of Ala537 in the variant simulations. Consequently, Pro541 disrupts the continuity of the secondary structure element, causing the α-helix to bend slightly in the variant 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.1631G>C	R544P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2				-16.905	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.762	Likely Pathogenic	4.70	Destabilizing	0.1	4.19	Destabilizing	4.45	Destabilizing	1.14	Destabilizing	-4.88	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.48	Pathogenic	0.05	Affected	3.37	35	0	-2	2.9	-59.07	192.0	123.8	0.1	0.0	-0.3	0.0	X	X						Potentially Pathogenic	Arg544 is located in the middle of an α-helix (res. Ala533-Val560). In the WT simulations, the guanidinium side chain of Arg544 forms a salt bridge with the carboxylate groups of Glu548 on the same α-helix, and with Glu651 and Glu656 on an opposing α-helix (res. Glu666-Asp644). In the variant simulations, the pyrrolidine side chain of Pro544 cannot form any of the salt bridges that Arg544 does in the WT, potentially weakening the tertiary structure assembly. Additionally, Pro544 lacks the amide group, and thus, unlike Arg544 in the WT, is unable to form a hydrogen bond with the carbonyl of Gln540. This disruption breaks the continuity of the secondary structure element, causing the α-helix to bend slightly in the variant simulations. These negative structural effects could be more pronounced during protein folding and are likely to be undermined in the MD simulations.
c.1976C>T	S659F (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.925	Likely Pathogenic	0.662	Likely Pathogenic	Likely Benign	0.194	Likely Benign	-0.81	Ambiguous	0.1	-0.25	Likely Benign	-0.53	Ambiguous	0.32	Likely Benign	-4.59	Deleterious	0.806	Possibly Damaging	0.171	Benign	3.39	Benign	0.05	Affected	3.38	28	-3	-2	3.6	60.10	221.3	-61.2	0.0	0.0	0.6	0.4						X		Potentially Benign	In the WT simulations, the hydroxyl group of Ser659, located in a kink in the middle of the long α-helix (res. Ser641-Glu666), forms a hydrogen bond with the carboxylate group of Glu656. However, the phenol ring of the Phe659 side chain cannot form a similar hydrogen bond. Instead, it interacts with the hydrophobic isopropyl side chain of Val555 from the opposing α-helix (res. Ala533-Val560). This residue swap may therefore cause issues during protein folding.
c.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.2050G>A	D684N (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.155	Likely Pathogenic	0.985	Likely Pathogenic	Likely Pathogenic	0.382	Likely Benign	1.47	Ambiguous	0.8	1.76	Ambiguous	1.62	Ambiguous	0.37	Likely Benign	-4.99	Deleterious	0.999	Probably Damaging	0.746	Possibly Damaging	3.39	Benign	0.01	Affected			2	1	0.0	-0.98
c.2050G>C	D684H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.194	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.613	Likely Pathogenic	3.36	Destabilizing	1.0	2.95	Destabilizing	3.16	Destabilizing	0.55	Ambiguous	-6.98	Deleterious	1.000	Probably Damaging	0.972	Probably Damaging	3.36	Benign	0.00	Affected	3.42	17	-1	1	0.3	22.05
c.1639T>C	C547R (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.967	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.900	Likely Pathogenic	7.76	Destabilizing	0.8	5.83	Destabilizing	6.80	Destabilizing	1.69	Destabilizing	-11.60	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	-1.33	Pathogenic	0.02	Affected	3.37	35	-4	-3	-7.0	53.05	267.4	-90.3	0.0	0.0	-0.1	0.1	X	X	X				X	Potentially Pathogenic	Cys547 is located in an α-helix (res. Ala533-Val560). The thiol side chain of Cys is situated in a hydrophobic inter-helix space, where it packs hydrophobically with other residues such as Ile626, Leu551, and Phe652. Additionally, the thiol side chain of Cys547 weakly hydrogen bonds with the carbonyl group of Leu543 in the same α-helix. In the variant simulations, the bulkier, positively charged guanidinium group of Arg547 must rotate out of the hydrophobic space. Consequently, it forms ionic interactions with the carboxylate groups of Glu548 in the same helix and Glu656 in the neighboring α-helix (res. Glu666-Asp644). This causes the two helices to slightly separate, significantly affecting the secondary structure integrity of the latter helix. These negative structural effects could be more pronounced during protein folding and are likely to be undermined in the MD simulations.
c.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.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.1658A>C	K553T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-15.328	Likely Pathogenic	0.990	Likely Pathogenic	Likely Pathogenic	0.761	Likely Pathogenic	1.06	Ambiguous	0.2	0.48	Likely Benign	0.77	Ambiguous	0.79	Ambiguous	-5.77	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.34	Pathogenic	0.14	Tolerated	3.37	35	0	-1	3.2	-27.07	218.2	-10.7	0.0	0.0	-0.2	0.5		X						Potentially Pathogenic	Lys533 is located on an α-helix (res. Ala533-Val560). In the WT simulations, Lys533 packs against Phe513, and its amino side chain occasionally forms an ionic interaction with the carboxylate group of Glu512 from an opposing α-helix (res. Gln503-Tyr518). In the variant simulations, Thr533 is unable to reproduce these interactions, potentially weakening the integrity of the tertiary structure. Additionally, Thr533 forms a hydrogen bond with the backbone carbonyl group of Leu549 in the same helix, which could potentially weaken the secondary structure. Regardless, the residue swap does not cause significant structural effects based on the simulations.
c.2060G>A	R687Q (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1				-10.002	Likely Pathogenic	0.575	Likely Pathogenic	Likely Benign	0.401	Likely Benign	0.92	Ambiguous	0.1	-0.37	Likely Benign	0.28	Likely Benign	1.55	Destabilizing	-3.37	Deleterious	1.000	Probably Damaging	0.844	Possibly Damaging	3.91	Benign	0.03	Affected	3.42	17	1	1	1.0	-28.06
c.2075T>A	L692Q (3D Viewer)	Likely Pathogenic	GAP	Pathogenic		1				-13.873	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.596	Likely Pathogenic	3.24	Destabilizing	0.1	3.27	Destabilizing	3.26	Destabilizing	2.76	Destabilizing	-5.98	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.06	Benign	0.00	Affected	3.42	17	-2	-2	-7.3	14.97
c.2086C>G	L696V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-11.909	Likely Pathogenic	0.745	Likely Pathogenic	Likely Benign	0.351	Likely Benign	2.35	Destabilizing	0.1	1.85	Ambiguous	2.10	Destabilizing	1.46	Destabilizing	-2.79	Deleterious	0.992	Probably Damaging	0.970	Probably Damaging	3.16	Benign	0.00	Affected	3.46	13	1	2	0.4	-14.03
c.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.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.1714T>C	W572R (3D Viewer)	Likely Pathogenic	GAP	Not provided		1				-17.511	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.894	Likely Pathogenic	4.84	Destabilizing	0.1	6.19	Destabilizing	5.52	Destabilizing	1.79	Destabilizing	-12.81	Deleterious					-1.25	Pathogenic	0.00	Affected	3.37	35	2	-3	-3.6	-30.03	312.6	-37.6	0.0	0.0	-1.0	0.0		X	X					Potentially Pathogenic	The indole ring of Trp572, located in an α-helix (res. Arg563-Glu578), lies in a hydrophobic inter-helix space, where it makes extensive hydrophobic interactions with nearby residues such as Met470, Phe569, Leu588, and Ile589. The guanidinium group of Arg572 is similarly sized to the tryptophan it replaced; however, it is also positively charged. In the variant simulations, Arg572 forms hydrogen bonds with other residues in the inter-helix space, such as Ser592 and the backbone carbonyl atom of Leu465. Additionally, Arg572 hydrophobically packs its carbon chain with surrounding residues such as Phe569 and Ile589.However, the introduced residue arginine is too hydrophilic and charged for the hydrophobic space, disrupting the hydrophobic packing of the inter-helix space. Indeed, in the second simulation, Arg572 successfully escapes the hydrophobic niche completely, causing the whole protein to partially unfold.Overall, the residue swap is highly likely to cause critical protein folding problems, as evidenced by the effects seen in the variant simulations.
c.1714T>G	W572G (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-17.692	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.900	Likely Pathogenic	6.57	Destabilizing	0.2	7.57	Destabilizing	7.07	Destabilizing	1.83	Destabilizing	-11.98	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.24	Pathogenic	0.00	Affected	3.37	35	-7	-2	0.5	-129.16	195.2	127.9	0.0	0.0	-1.0	0.0							X	Potentially Pathogenic	The introduced residue Gly572, 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 Gly572 essentially lacks a side chain altogether. Although not observed in the simulations, the residue swap could also weaken the integrity of the helix (res. Arg563-Glu578), as glycine is known as an “α-helix breaker.” 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.2111G>C	S704T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.930	Likely Benign	0.265	Likely Benign	Likely Benign	0.071	Likely Benign	0.80	Ambiguous	0.0	0.15	Likely Benign	0.48	Likely Benign	0.29	Likely Benign	-1.72	Neutral	0.525	Possibly Damaging	0.107	Benign	3.45	Benign	0.07	Tolerated	3.47	10	1	1	0.1	14.03	201.7	-18.0	0.0	0.0	-0.2	0.7						X		Potentially Benign	Ser704 is located at the end and outer surface of an α-helix (res. Thr704-Gly712), which is connected via a tight turn or loop to another α-helix (res. Asp684-Gln702). The hydroxyl side chain of Ser704 occasionally forms a hydrogen bond with the amide group of Ala707. Similarly, in the variant simulations, the hydroxyl side chain of Thr704 forms hydrogen bonds with the amide groups of Ala707 and Leu708. Thus, the residue swap does not cause any apparent structural change.
c.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.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.1717C>T	R573W (3D Viewer)	Likely Pathogenic	GAP	Conflicting		8				-14.078	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.758	Likely Pathogenic	2.37	Destabilizing	0.7	0.57	Ambiguous	1.47	Ambiguous	0.88	Ambiguous	-6.94	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	-1.48	Pathogenic	0.00	Affected	3.37	35	2	-3	3.6	30.03	257.6	39.0	0.1	0.0	0.2	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, the indole ring of the Trp573 side chain is unable to maintain the same level of coordination as the positively charged Arg573 side chain. Indeed, Trp573 is seen hydrogen bonding only briefly with the carboxylate group of Glu582. 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>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.2186A>G	N729S (3D Viewer)	Likely Benign	GAP	Uncertain		1				-1.578	Likely Benign	0.066	Likely Benign	Likely Benign	0.063	Likely Benign	0.14	Likely Benign	0.1	1.34	Ambiguous	0.74	Ambiguous	-0.36	Likely Benign	-0.42	Neutral	0.221	Benign	0.027	Benign	3.38	Benign	0.93	Tolerated	3.59	7	1	1	2.7	-27.03
c.2195G>C	R732T			Uncertain		1				-8.545	Likely Pathogenic	0.434	Ambiguous	Likely Benign	0.075	Likely Benign										-1.96	Neutral	0.999	Probably Damaging	0.892	Possibly Damaging	2.59	Benign	0.12	Tolerated	3.59	7	-1	-1	3.8	-55.08
c.2210A>C	Q737P	Likely Benign		Uncertain		1				-2.407	Likely Benign	0.054	Likely Benign	Likely Benign	0.154	Likely Benign										-1.22	Neutral	0.005	Benign	0.013	Benign	2.78	Benign	0.04	Affected	4.07	3	-1	0	1.9	-31.01
c.2215G>C	E739Q	Likely Benign		Uncertain		1				-2.846	Likely Benign	0.161	Likely Benign	Likely Benign	0.071	Likely Benign										-1.06	Neutral	0.801	Possibly Damaging	0.339	Benign	2.57	Benign	0.00	Affected	4.32	2	2	2	0.0	-0.98
c.2216A>T	E739V	Likely Benign		Uncertain		1				-3.136	Likely Benign	0.274	Likely Benign	Likely Benign	0.085	Likely Benign										-1.86	Neutral	0.891	Possibly Damaging	0.575	Possibly Damaging	2.47	Pathogenic	0.00	Affected	4.32	2	-2	-2	7.7	-29.98
c.1741C>T	R581W (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2				-12.855	Likely Pathogenic	0.920	Likely Pathogenic	Ambiguous	0.678	Likely Pathogenic	1.32	Ambiguous	0.1	-0.32	Likely Benign	0.50	Ambiguous	0.68	Ambiguous	-6.79	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	-1.37	Pathogenic	0.01	Affected	3.37	34	2	-3	3.6	30.03	257.8	36.0	0.1	0.1	0.1	0.3	X	X						Potentially Pathogenic	Arg581 is located on a short α-α loop between two α helices (res. Arg563-Glu578 and res. Glu582-Ser604). In the WT simulations, the guanidinium group of Arg581 forms salt bridges with the carboxylate groups of Asp583 within the same helix, as well as with Glu478 and/or Glu480 in a slightly α-helical loop (res. Glu478-Thr488) preceding another α helix (res. Ala461-Phe476).In the variant simulations, the neutral indole ring of the Trp581 side chain cannot form any of these salt bridges. Instead, it packs hydrophobically against Met477 and Ile587 without forming any direct hydrogen bonds. The tendency of the loop (res. Asp477-Thr488) to acquire an α-helical structure seems to marginally increase, potentially due to Trp581's inability to coordinate stable hydrogen bonds with the loop residues (e.g., Glu478-Arg581 salt bridge). Additionally, the residue swap could weaken the tertiary structure assembly and negatively affect the overall protein folding process.
c.2217G>C	E739D	Likely Benign		Uncertain		1				-3.369	Likely Benign	0.062	Likely Benign	Likely Benign	0.097	Likely Benign										-0.49	Neutral	0.002	Benign	0.005	Benign	2.59	Benign	0.00	Affected			3	2	0.0	-14.03
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.1760G>C	R587T (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.697	Likely Pathogenic	0.784	Likely Pathogenic	Likely Benign	0.603	Likely Pathogenic	1.14	Ambiguous	0.2	0.74	Ambiguous	0.94	Ambiguous	0.98	Ambiguous	-4.71	Deleterious	0.998	Probably Damaging	0.847	Possibly Damaging	-1.19	Pathogenic	0.08	Tolerated	3.37	35	-1	-1	3.8	-55.08	227.2	87.4	0.0	0.0	0.5	0.1		X						Potentially Pathogenic	The guanidinium group of Arg587, located on an α helix (res. Glu582-Met603), is constantly rotating and breaking/forming multiple hydrogen bonds and/or salt bridges at the surface intersection of α helices in the WT simulations. The positively charged Arg587 side chain can form a salt bridge with either the carboxylate group of Asp583 or Asp586 in the same helix, or with Glu480 on the opposing short helical loop structure (res. Glu480-Leu482).Importantly, the Arg587 side chain also hydrogen bonds with the backbone carbonyl groups of Ala634 and Asn635, as well as the carboxamide group of Asn635 at the end of another α helix (res. Asp616-Phe636). However, in the variant simulations, the neutral hydroxyl group of the Thr587 side chain is unable to form these salt bridges. Due to its smaller size, it also does not form the hydrogen bonds that the Arg587 side chain could. Instead, the hydroxyl group of Thr587 hydrogen bonds with the backbone carbonyl group of Asp583, which could weaken the integrity of the α helix, although this is not observed in the simulations.Overall, the residue swap could weaken the tertiary structure assembly and negatively affect the overall protein folding process.
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.1767C>G	I589M (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.225	Likely Pathogenic	0.926	Likely Pathogenic	Ambiguous	0.830	Likely Pathogenic	0.74	Ambiguous	0.2	1.54	Ambiguous	1.14	Ambiguous	1.33	Destabilizing	-2.99	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.94	Pathogenic	0.00	Affected	3.37	35	2	1	-2.6	18.03	267.6	-24.5	0.0	0.0	-0.1	0.1						X		Potentially Benign	A hydrophobic residue, Ile589, located in an α helix (res. Glu582-Met603), is swapped for another hydrophobic residue, methionine. The sec-butyl hydrocarbon side chain of Ile589 packs favourably with multiple residues in the inter-helix hydrophobic space (e.g., Phe569, Ile667, and Leu664).Although the S-methyl thioether group of the Met589 side chain in the variant is longer than the branched side chain of isoleucine, it stacks favourably with the aromatic phenol ring. Additionally, the polar sulphur atom forms a weak hydrogen bond with the guanidinium group of Arg573, which in turn forms a salt bridge with the carboxylate group of Asp586.Overall, the hydrophobic packing in the inter-helix space does not appear to be disrupted in the variant simulations.
c.2249G>A	G750E			Uncertain		1				-2.618	Likely Benign	0.413	Ambiguous	Likely Benign	0.146	Likely Benign										-2.27	Neutral	1.000	Probably Damaging	0.982	Probably Damaging	2.49	Pathogenic	0.01	Affected	3.99	5	0	-2	-3.1	72.06
c.2270G>C	G757A	Likely Benign		Uncertain		1				-2.626	Likely Benign	0.091	Likely Benign	Likely Benign	0.066	Likely Benign										-0.45	Neutral	0.267	Benign	0.127	Benign	2.73	Benign	0.35	Tolerated			1	0	2.2	14.03
c.1771G>C	A591P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.479	Likely Pathogenic	0.991	Likely Pathogenic	Likely Pathogenic	0.404	Likely Benign	3.78	Destabilizing	0.3	7.29	Destabilizing	5.54	Destabilizing	1.45	Destabilizing	-4.41	Deleterious	0.995	Probably Damaging	0.853	Possibly Damaging	3.35	Benign	0.01	Affected	3.37	35	1	-1	-3.4	26.04	191.5	-10.1	0.2	0.1	0.4	0.1	X							Potentially Pathogenic	The methyl group of the Ala591 side chain, located in the middle of an α helix (res. Glu582-Met603), packs against hydrophobic residues (e.g., Ile483, Phe484) of an opposing partially helical loop (res. Phe476-Asn487).In the variant simulations, Pro591 lacks a free backbone amide group and, therefore, cannot form a hydrogen bond with the backbone carbonyl of Arg587 as Ala591 does in the WT. This notably weakens the α helix integrity and compromises the continuity of the helix. In reality, the effect on the structure during protein folding could be more severe.
c.1778T>A	L593H (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.504	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.812	Likely Pathogenic	2.52	Destabilizing	0.2	2.32	Destabilizing	2.42	Destabilizing	2.75	Destabilizing	-6.77	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.77	Benign	0.00	Affected	3.37	35	-2	-3	-7.0	23.98	222.0	20.7	0.0	0.0	0.2	0.0		X					X	Potentially Pathogenic	The iso-propyl side chain of Leu593, located in an α helix (res. Glu582-Met603), packs favourably with multiple hydrophobic residues in the inter-helix space (e.g., Leu598, Ile589, Phe594, Phe561).In the variant simulations, His593 retains a similar packing arrangement via its aromatic imidazole ring. However, the polar nitrogen atoms introduce hydrogen bond donors and acceptors into the previously hydrophobic space. The epsilon protonated nitrogen of His593 forms a stable hydrogen bond with the phenol group of the Tyr505 side chain in an α helix (res. Gln503-Tyr518).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.2291A>G	N764S	Likely Benign		Benign		1				-3.149	Likely Benign	0.159	Likely Benign	Likely Benign	0.058	Likely Benign										-0.84	Neutral	0.992	Probably Damaging	0.846	Possibly Damaging	2.65	Benign	0.61	Tolerated	3.64	6	1	1	2.7	-27.03
c.2294G>A	S765N	Likely Benign		Uncertain		1				-5.098	Likely Benign	0.378	Ambiguous	Likely Benign	0.094	Likely Benign										-0.94	Neutral	0.985	Probably Damaging	0.950	Probably Damaging	4.11	Benign	0.06	Tolerated	3.64	6	1	1	-2.7	27.03
c.2299A>G	I767V	Likely Benign		Uncertain		1				-2.791	Likely Benign	0.064	Likely Benign	Likely Benign	0.096	Likely Benign										0.10	Neutral	0.072	Benign	0.029	Benign	4.21	Benign	1.00	Tolerated	3.64	6	4	3	-0.3	-14.03
c.1787G>T	R596L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.197	Likely Pathogenic	0.992	Likely Pathogenic	Likely Pathogenic	0.756	Likely Pathogenic	1.51	Ambiguous	0.3	-0.58	Ambiguous	0.47	Likely Benign	-0.02	Likely Benign	-6.97	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.45	Pathogenic	0.00	Affected	3.37	35	-3	-2	8.3	-43.03	234.2	63.4	-0.1	0.0	-0.5	0.6		X		X				Potentially Pathogenic	The guanidinium group of Arg596, located in an α helix (res. Glu582-Met603), forms a salt bridge with the carboxylate group of Glu495 from another α helix (res. Leu489-Glu519). In the WT simulations, the side chain of Arg596 hydrogen bonds with the backbone carbonyl groups of Asn487, Glu486, Arg485, and Phe484. Additionally, Arg596 can hydrogen bond with the carboxamide group of the Asn487 side chain on an opposing loop that links two α helices (res. Ala461-Arg475, res. Leu489-Glu519).However, in the variant simulations, the branched hydrocarbon side chain of Leu596 cannot form any of the hydrogen bonds or salt bridges maintained by the considerably bulkier and positively charged Arg596 side chain. Instead, Leu596 packs hydrophobically with the phenyl ring of Phe484 in the linker loop or residues from the opposing helix (e.g., Ile494, Thr491).Thus, the residue swap could affect the tertiary structure assembly more profoundly than observed in the simulations. Notably, Arg596 plays a key role in positioning the aforementioned loop, which is crucial for the placement of the “arginine finger” or the Arg485 side chain during RasGTPase activation.	10.1016/j.ajhg.2020.11.011
c.1802C>A	A601E (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2				-16.752	Likely Pathogenic	0.992	Likely Pathogenic	Likely Pathogenic	0.588	Likely Pathogenic	6.68	Destabilizing	0.8	5.76	Destabilizing	6.22	Destabilizing	1.24	Destabilizing	-4.98	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.54	Benign	0.00	Affected	3.37	35	0	-1	-5.3	58.04	240.0	-82.3	0.0	0.0	0.7	0.1		X	X				X	Potentially Pathogenic	The methyl side chain of Ala601, located on an α helix (res. Glu582-Met603), packs hydrophobically against other hydrophobic residues in the inter-helix space (e.g., Phe597, Leu598, Leu506, Phe608).In the variant simulations, the carboxylate group of Glu601 faces the inter-helix space and is forced to shift slightly away from the hydrophobic niche. Additionally, in two of the simulations, Glu601 forms a salt bridge with Arg499, causing the otherwise stable salt bridge between Arg499 and Glu496 at the outer surface of an α helix (res. Leu489-Glu519) to break due to the residue swap.These effects suggest that the protein folding process could be seriously affected. Moreover, due to its location at the GAP-Ras interface, it could also impact the complex formation with the GTPase.
c.1802C>T	A601V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.447	Likely Pathogenic	0.853	Likely Pathogenic	Ambiguous	0.535	Likely Pathogenic	1.64	Ambiguous	0.1	0.35	Likely Benign	1.00	Ambiguous	0.81	Ambiguous	-3.98	Deleterious	1.000	Probably Damaging	0.989	Probably Damaging	2.74	Benign	0.03	Affected	3.37	35	0	0	2.4	28.05	228.5	-45.5	0.0	0.0	0.4	0.5						X		Potentially Benign	The methyl side chain of Ala601, located on an α helix (res. Glu582-Met603), packs hydrophobically against other hydrophobic residues in the inter-helix space (e.g., Phe597, Leu598, Leu506, Phe608).In the variant simulations, Val601, which has similar size and physicochemical properties to alanine, resides in the inter-helix hydrophobic space in a similar manner to Ala601 in the WT, causing no apparent negative effect on the protein structure. However, the effect of the residue swap on the SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.2300T>C	I767T	Likely Benign		Uncertain		1				-3.749	Likely Benign	0.252	Likely Benign	Likely Benign	0.138	Likely Benign										-0.78	Neutral	0.625	Possibly Damaging	0.249	Benign	4.12	Benign	0.46	Tolerated	3.64	6	0	-1	-5.2	-12.05
c.2302G>T	D768Y	Likely Pathogenic		Uncertain		1	6-33442460-G-T			-9.866	Likely Pathogenic	0.824	Likely Pathogenic	Ambiguous	0.234	Likely Benign										-2.86	Deleterious	0.989	Probably Damaging	0.806	Possibly Damaging	4.01	Benign	0.07	Tolerated	3.64	6	-4	-3	2.2	48.09
c.2305C>T	L769F	Likely Benign		Uncertain		1				-5.044	Likely Benign	0.146	Likely Benign	Likely Benign	0.060	Likely Benign										-0.89	Neutral	0.925	Possibly Damaging	0.510	Possibly Damaging	3.94	Benign	0.02	Affected			2	0	-1.0	34.02
c.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.233G>T	R78L	Likely Benign		Uncertain		1				-3.389	Likely Benign	0.635	Likely Pathogenic	Likely Benign	0.062	Likely Benign										-1.59	Neutral	0.385	Benign	0.021	Benign	3.84	Benign	0.00	Affected			-3	-2	8.3	-43.03
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.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.1813C>T	P605S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-10.830	Likely Pathogenic	0.987	Likely Pathogenic	Likely Pathogenic	0.718	Likely Pathogenic	3.40	Destabilizing	0.1	3.34	Destabilizing	3.37	Destabilizing	1.00	Destabilizing	-7.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	0.70	Pathogenic	0.00	Affected	3.37	35	1	-1	0.8	-10.04	213.8	-15.4	-0.3	0.2	0.2	0.1				X			X	Potentially Pathogenic	Pro605 is located in a short turn between an α helix (res. Glu582-Met603) and a short α helical section (res. Ser606-Phe608). The pyrrolidine side chain of Pro605 packs hydrophobically with nearby hydrophobic residues (e.g., Ile514, Leu623, Leu610) in the inter-helix space. Additionally, proline lacks a free backbone amide group, which breaks the α helix and facilitates the turn in the WT structure.In the variant simulations, the hydroxyl side chain of Ser605 forms hydrogen bonds with the backbone carbonyl groups of Ala601 and Ile602. Importantly, the helix end is more stable than with Pro605 in the WT. Indeed, proline is a more effective secondary structure breaker compared to serine.Thus, the residue swap could have a more profound effect on the actual folding process, for example, by preventing the bending at the α helix end, than what the simulations suggest. Moreover, due to its location at the GAP-Ras interface, the residue swap could affect the GAP-Ras association.
c.1814C>G	P605R (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-13.745	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.845	Likely Pathogenic	8.71	Destabilizing	2.5	6.46	Destabilizing	7.59	Destabilizing	0.92	Ambiguous	-8.95	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	0.69	Pathogenic	0.00	Affected	3.37	35	0	-2	-2.9	59.07	281.7	-118.1	-0.2	0.0	0.5	0.1		X	X	X			X	Potentially Pathogenic	Pro605 is located in a short turn between an α helix (res. Glu582-Met603) and a short α helical section (res. Ser606-Phe608). The pyrrolidine side chain of Pro605 packs hydrophobically with nearby hydrophobic residues (e.g., Ile514, Leu623, Leu610) in the inter-helix space. Additionally, proline lacks a free backbone amide group, which breaks the α helix and facilitates the turn in the WT structure.In the variant simulations, the guanidinium side chain of Arg605 is bulkier than proline, and its positively charged guanidinium group faces mostly hydrophobic residues (e.g., Ile514, Leu623, Leu610). As a result, it needs to rotate away from the hydrophobic niche. The residue swap could have a more profound effect on the actual folding process, for example, by preventing the bending at the α helix end.Moreover, due to its location at the GAP-Ras interface, the residue swap could affect the GAP-Ras association.
c.2369C>G	T790S	Likely Benign	SH3-binding motif	Uncertain		1				-3.914	Likely Benign	0.123	Likely Benign	Likely Benign	0.134	Likely Benign										-1.83	Neutral	0.997	Probably Damaging	0.989	Probably Damaging	2.39	Pathogenic	0.33	Tolerated	3.64	6	1	1	-0.1	-14.03
c.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.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.2401G>A	G801S	Likely Benign	SH3-binding motif	Uncertain		1				-3.665	Likely Benign	0.087	Likely Benign	Likely Benign	0.039	Likely Benign										-0.41	Neutral	0.009	Benign	0.019	Benign	2.76	Benign	0.48	Tolerated	4.32	2	0	1	-0.4	30.03
c.2408A>G	K803R	Likely Benign	SH3-binding motif	Uncertain		1				-2.281	Likely Benign	0.097	Likely Benign	Likely Benign	0.018	Likely Benign										-1.52	Neutral	0.103	Benign	0.038	Benign	2.38	Pathogenic	0.00	Affected	3.77	5	3	2	-0.6	28.01
c.2414T>C	L805P		SH3-binding motif	Uncertain		1				-4.661	Likely Benign	0.444	Ambiguous	Likely Benign	0.272	Likely Benign										-3.40	Deleterious	0.975	Probably Damaging	0.767	Possibly Damaging	2.36	Pathogenic	0.00	Affected	3.77	5	-3	-3	-5.4	-16.04
c.2420A>T	Y807F	Likely Benign	SH3-binding motif	Uncertain		1				-3.667	Likely Benign	0.073	Likely Benign	Likely Benign	0.057	Likely Benign										0.14	Neutral	0.012	Benign	0.022	Benign	2.92	Benign	0.98	Tolerated	3.77	5	7	3	4.1	-16.00
c.1947G>C	M649I (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-9.361	Likely Pathogenic	0.995	Likely Pathogenic	Likely Pathogenic	0.449	Likely Benign	2.42	Destabilizing	0.2	1.96	Ambiguous	2.19	Destabilizing	1.01	Destabilizing	-3.99	Deleterious	0.672	Possibly Damaging	0.093	Benign	3.40	Benign	0.02	Affected	3.38	27	2	1	2.6	-18.03	243.7	21.5	0.0	0.1	0.0	0.1							X	Potentially Benign	The thioether side chain of Met649, located on an α helix (res. Ser641-Glu666), bridges Phe652, Phe648, and Phe639 in an inter-helix hydrophobic cavity in the WT simulations. In the variant simulations, the sec-butyl side chain of Ile649 maintains hydrophobic interactions with nearby residues, with no significant effects on the protein structure.However, methionine is known as a bridging motif for aromatic residues, and these Met-aromatic interactions are lost in the variant. Indeed, in the second variant simulation,the bridging of Phe652, Phe648 and Phe639 is completely lost. In reality, the effect could be more severe on the structure during the protein folding.
c.1998G>C	E666D (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-8.820	Likely Pathogenic	0.704	Likely Pathogenic	Likely Benign	0.197	Likely Benign	0.88	Ambiguous	0.0	0.37	Likely Benign	0.63	Ambiguous	1.05	Destabilizing	-2.69	Deleterious	0.992	Probably Damaging	0.603	Possibly Damaging	3.43	Benign	0.06	Tolerated	3.38	28	3	2	0.0	-14.03	237.2	16.5	0.0	0.0	-0.3	0.1		X						Potentially Pathogenic	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 WT simulations. In the variant simulations, the shorter side chain of Asp666 cannot maintain these interactions as efficiently as Glu666 in the WT, resulting in a less coordinated hydrogen-bond network.
c.2443C>A	R815S		SH3-binding motif	Benign		1				-7.324	In-Between	0.950	Likely Pathogenic	Ambiguous	0.138	Likely Benign										-1.86	Neutral	0.999	Probably Damaging	0.997	Probably Damaging	2.67	Benign	0.02	Affected			0	-1	3.7	-69.11
c.2443C>G	R815G		SH3-binding motif	Uncertain		1				-7.983	In-Between	0.854	Likely Pathogenic	Ambiguous	0.146	Likely Benign										-3.22	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.62	Benign	0.02	Affected	4.32	4	-3	-2	4.1	-99.14
c.2444G>T	R815L	Likely Pathogenic	SH3-binding motif	Uncertain		1				-8.546	Likely Pathogenic	0.865	Likely Pathogenic	Ambiguous	0.175	Likely Benign										-3.06	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.63	Benign	0.03	Affected	4.32	4	-2	-3	8.3	-43.03
c.2458T>A	Y820N			Uncertain		1				-9.032	Likely Pathogenic	0.842	Likely Pathogenic	Ambiguous	0.143	Likely Benign										-1.53	Neutral	0.999	Probably Damaging	0.977	Probably Damaging	2.74	Benign	0.20	Tolerated			-2	-2	-2.2	-49.07
c.2459A>G	Y820C	Likely Pathogenic		Uncertain		1				-8.797	Likely Pathogenic	0.744	Likely Pathogenic	Likely Benign	0.113	Likely Benign										-3.16	Deleterious	1.000	Probably Damaging	0.983	Probably Damaging	2.68	Benign	0.06	Tolerated	3.77	5	0	-2	3.8	-60.04
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.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.2015C>A	T672K (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-12.192	Likely Pathogenic	0.698	Likely Pathogenic	Likely Benign	0.065	Likely Benign	0.20	Likely Benign	0.5	1.21	Ambiguous	0.71	Ambiguous	0.72	Ambiguous	-4.31	Deleterious	0.745	Possibly Damaging	0.051	Benign	3.40	Benign	0.07	Tolerated	3.40	25	0	-1	-3.2	27.07	195.1	7.0	0.4	0.7	0.4	0.1		X				X		Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop itself, such as Lys566, Glu666, and Asn669. In the variant simulations, Lys672 can only form a hydrogen bond with the amino group of the Lys566 side chain via its backbone carbonyl group. Consequently, it cannot maintain the Lys566-Glu666 salt bridge through hydrogen bonding. However, the amino group of Lys periodically forms a salt bridge with the carboxylate group of Glu666, which prevents a drastic disruption of the hydrogen-bond network that keeps the loop close to the helices.
c.249A>T	R83S	Likely Benign		Uncertain		1				-2.550	Likely Benign	0.999	Likely Pathogenic	Likely Pathogenic	0.094	Likely Benign										-1.87	Neutral	0.909	Possibly Damaging	0.587	Possibly Damaging	3.19	Benign	0.00	Affected	4.32	1	0	-1	3.7	-69.11
c.2502G>C	M834I	Likely Benign		Uncertain		1				-3.377	Likely Benign	0.291	Likely Benign	Likely Benign	0.055	Likely Benign										-1.21	Neutral	0.026	Benign	0.009	Benign	2.56	Benign	0.00	Affected	4.32	4	1	2	2.6	-18.03
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.250C>G	R84G			Uncertain		1				-6.627	Likely Benign	0.989	Likely Pathogenic	Likely Pathogenic	0.139	Likely Benign										-2.64	Deleterious	0.962	Probably Damaging	0.726	Possibly Damaging	3.68	Benign	0.00	Affected	4.32	1	-3	-2	4.1	-99.14
c.2514C>A	N838K	Likely Pathogenic		Uncertain		2				-8.470	Likely Pathogenic	0.862	Likely Pathogenic	Ambiguous	0.097	Likely Benign										-2.78	Deleterious	0.997	Probably Damaging	0.995	Probably Damaging	2.69	Benign	0.16	Tolerated	3.77	5	1	0	-0.4	14.07
c.2518A>T	S840C	Likely Pathogenic		Uncertain		1				-8.799	Likely Pathogenic	0.904	Likely Pathogenic	Ambiguous	0.376	Likely Benign										-3.96	Deleterious	0.999	Probably Damaging	0.975	Probably Damaging	1.50	Pathogenic	0.00	Affected	3.77	5	0	-1	3.3	16.06
c.2068T>C	S690P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.568	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.431	Likely Benign	4.84	Destabilizing	0.3	4.40	Destabilizing	4.62	Destabilizing	1.42	Destabilizing	-4.77	Deleterious	0.998	Probably Damaging	0.790	Possibly Damaging	3.44	Benign	0.01	Affected	3.42	17	1	-1	-0.8	10.04	207.5	15.1	0.1	0.0	-0.1	0.2	X	X						Potentially Pathogenic	The hydroxyl side chain of Ser690, located in an α-helix (res. Leu696-Leu685), forms a hydrogen bond with the backbone carbonyl group of Ser410 in an anti-parallel β-sheet of the C2 domain (res. Ile411-Ala399). In the variant simulations, the pyrrolidine side chain of Pro690 cannot form hydrogen bonds with the C2 domain residue, resulting in the loss of this inter-domain connection. Additionally, prolines lack a free amide group necessary for hydrogen bonding with the carbonyl group of Gly686, introducing a slight bend in the α-helix and compromising its integrity.
c.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.2075T>C	L692P (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-16.447	Likely Pathogenic	1.000	Likely Pathogenic	Likely Pathogenic	0.668	Likely Pathogenic	9.19	Destabilizing	0.1	13.20	Destabilizing	11.20	Destabilizing	1.69	Destabilizing	-6.98	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	3.06	Benign	0.00	Affected	3.42	17	-3	-3	-5.4	-16.04	186.2	62.8	-0.2	0.1	-0.7	0.3	X							Potentially Pathogenic	The isobutyl side chain of Leu692, located in the middle of an α-helix (res. Leu685-Gln702), engages in hydrophobic packing with nearby residues (e.g., Leu441, Leu431, Leu696) in the inter-helix space. Prolines lack a free amide group necessary for hydrogen bonding with the carbonyl group of Glu688 in the same manner as Leu692 in the WT. Consequently, the residue swap with proline disrupts the continuity of the secondary structure element in the variant simulations. Additionally, the side chain of Pro692 is not as optimal as Leu692 for hydrophobic packing in the inter-helix space.
c.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.2548G>A	G850R	Likely Benign		Uncertain		1				-5.082	Likely Benign	0.398	Ambiguous	Likely Benign	0.194	Likely Benign										-0.07	Neutral	0.010	Benign	0.010	Benign	4.30	Benign	0.01	Affected	3.77	5	-3	-2	-4.1	99.14
c.2557G>C	G853R	Likely Benign		Uncertain		1				-4.749	Likely Benign	0.366	Ambiguous	Likely Benign	0.091	Likely Benign										-1.27	Neutral	0.846	Possibly Damaging	0.624	Possibly Damaging	4.18	Benign	0.00	Affected			-3	-2	-4.1	99.14
c.256G>A	V86I	Likely Benign		Uncertain		1				-4.726	Likely Benign	0.338	Likely Benign	Likely Benign	0.076	Likely Benign										-0.31	Neutral	0.267	Benign	0.097	Benign	3.94	Benign	0.00	Affected	4.32	1	4	3	0.3	14.03
c.2105A>G	Q702R (3D Viewer)		GAP	Uncertain		1				-7.894	In-Between	0.348	Ambiguous	Likely Benign	0.294	Likely Benign	-0.31	Likely Benign	0.1	0.63	Ambiguous	0.16	Likely Benign	0.13	Likely Benign	-3.14	Deleterious	0.909	Possibly Damaging	0.889	Possibly Damaging	3.43	Benign	0.02	Affected	3.47	10	1	1	-1.0	28.06	270.3	-52.9	0.0	0.0	0.0	0.1		X						Potentially Pathogenic	The carboxamide side chain of Gln702 is located at the end and outer surface of an α-helix (res. Leu685-Gln702), where it does not directly form hydrogen bonds with any residues in the WT simulations. In the variant simulations, the positively charged guanidinium group of Arg702 forms a salt bridge with the negatively charged carboxylate group of Glu698 on the same helix and/or hydrogen bonds with the backbone carbonyl group of Ala438 on an opposite α-helix (res. Tyr428-Glu436). Consequently, the residue swap could strengthen the tertiary structure assembly, which could have either positive or negative effects on its function.
c.2116G>A	E706K (3D Viewer)		GAP	Uncertain		1				-10.519	Likely Pathogenic	0.833	Likely Pathogenic	Ambiguous	0.080	Likely Benign	1.17	Ambiguous	0.1	0.51	Ambiguous	0.84	Ambiguous	0.08	Likely Benign	-1.51	Neutral	0.345	Benign	0.028	Benign	4.15	Benign	0.52	Tolerated	3.47	10	0	1	-0.4	-0.94	187.1	49.2	0.0	0.0	0.4	0.1						X		Uncertain	The carboxylate side chain of Glu706, located at the end and outer surface of an α-helix (res. Thr704-Gly712), forms a salt bridge with Lys710 and a hydrogen bond with its own backbone amino group at the helix end in the WT simulations. Although Lys706 is unable to make these transient interactions in the variant simulations, there is no apparent negative effect on the protein structure due to the residue swap. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2162T>G	I721S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.032	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.466	Likely Benign	3.91	Destabilizing	0.1	3.96	Destabilizing	3.94	Destabilizing	2.28	Destabilizing	-5.26	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.21	Pathogenic	0.00	Affected	3.50	9	-1	-2	-5.3	-26.08	203.3	49.3	-0.1	0.0	-1.1	0.0							X	Uncertain	The sec-butyl side chain of Ile721, located on an α-helix (res. Leu714-Arg726), engages in hydrophobic packing with other residues in the hydrophobic inter-helix space, such as Phe420, Tyr417, His693, and Leu717. In the variant simulations, the hydroxyl side chain of Ser721 forms hydrogen bonds with nearby residues, such as Leu717 and His693. Although no major structural changes are observed during the variant simulations, the hydrophilic residue Ser721 could disrupt the hydrophobic packing during folding. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2608C>G	L870V	Likely Benign		Uncertain		1				-4.123	Likely Benign	0.300	Likely Benign	Likely Benign	0.111	Likely Benign										-1.19	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.64	Benign	0.12	Tolerated	3.88	3	2	1	0.4	-14.03
c.2627C>T	S876L			Uncertain		2				-5.856	Likely Benign	0.489	Ambiguous	Likely Benign	0.249	Likely Benign										-3.56	Deleterious	0.998	Probably Damaging	0.992	Probably Damaging	2.57	Benign	0.05	Affected	3.77	5	-2	-3	4.6	26.08
c.2632A>G	T878A	Likely Benign		Uncertain		1				-2.154	Likely Benign	0.081	Likely Benign	Likely Benign	0.088	Likely Benign										-0.67	Neutral	0.003	Benign	0.006	Benign	2.73	Benign	0.18	Tolerated	3.77	5	1	0	2.5	-30.03