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.1150G>A	G384S (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438055-G-A	1	6.22e-7	-5.243	Likely Benign	0.090	Likely Benign	Likely Benign	0.315	Likely Benign	1.92	Ambiguous	0.2	1.66	Ambiguous	1.79	Ambiguous	0.19	Likely Benign	-0.67	Neutral	0.980	Probably Damaging	0.968	Probably Damaging	1.33	Pathogenic	0.04	Affected	4.32	2	1	0	-0.4	30.03	202.4	-49.8	0.5	1.0	-0.2	0.0											Uncertain	Gly384 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycines, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Ser384 is potentially tolerated in the Ω loop, although the hydroxyl group of Ser384 forms various hydrogen bonds with several other loop residues in the variant simulations. However, since the effects on Gly-rich Ω loop dynamics can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.1153T>C	S385P (3D Viewer)	Likely Benign	C2	Uncertain		1	6-33438058-T-C			-5.431	Likely Benign	0.123	Likely Benign	Likely Benign	0.385	Likely Benign	0.91	Ambiguous	0.6	-0.90	Ambiguous	0.01	Likely Benign	0.19	Likely Benign	-0.26	Neutral	0.676	Possibly Damaging	0.693	Possibly Damaging	4.63	Benign	0.04	Affected	4.32	3	1	-1	-0.8	10.04	210.3	18.5	1.8	0.9	0.3	0.0								Uncertain	Ser385 is located in the Gly-rich Ω loop (res. Pro364-Pro398) between two anti-parallel β sheet strands (res. Thr359-Pro364, res. Ala399-Ile411). Because the Ω loop is assumed to directly interact with the membrane, it moves arbitrarily throughout the WT solvent simulations. The Ω loop potentially plays a crucial role in the SynGAP-membrane complex association, stability, and dynamics. However, this aspect cannot be fully addressed through solvent simulations alone.Ω loops are known to play major roles in protein functions that require flexibility, and so they are rich in glycine residues, prolines, and, to a lesser extent, small hydrophilic residues to ensure maximum flexibility. Thus, the variant’s Pro385 is potentially tolerated in the Ω loop. However, since the effects on Gly-rich Ω loop dynamics can only be well studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.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.1404G>A	M468I (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438436-G-A	1	6.20e-7	-8.583	Likely Pathogenic	0.907	Likely Pathogenic	Ambiguous	0.508	Likely Pathogenic	2.53	Destabilizing	0.2	1.89	Ambiguous	2.21	Destabilizing	0.37	Likely Benign	-1.06	Neutral	0.748	Possibly Damaging	0.886	Possibly Damaging	-1.10	Pathogenic	0.07	Tolerated	3.37	31	1	2	2.6	-18.03
c.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.140G>A	R47Q	Likely Benign		Likely Benign		1	6-33423549-G-A	4	2.48e-6	-4.989	Likely Benign	0.347	Ambiguous	Likely Benign	0.096	Likely Benign										-0.57	Neutral	0.829	Possibly Damaging	0.614	Possibly Damaging	4.12	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.1436G>A	R479Q (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33438468-G-A	7	4.34e-6	-7.109	In-Between	0.259	Likely Benign	Likely Benign	0.191	Likely Benign	0.54	Ambiguous	0.1	0.57	Ambiguous	0.56	Ambiguous	0.49	Likely Benign	-1.16	Neutral	1.000	Probably Damaging	0.991	Probably Damaging	3.42	Benign	0.31	Tolerated	3.39	32	1	1	1.0	-28.06
c.1195G>A	A399T (3D Viewer)	Likely Benign	C2	Benign		1				-5.236	Likely Benign	0.114	Likely Benign	Likely Benign	0.272	Likely Benign	1.24	Ambiguous	0.1	0.91	Ambiguous	1.08	Ambiguous	0.49	Likely Benign	-0.40	Neutral	0.131	Benign	0.039	Benign	5.41	Benign	0.69	Tolerated	3.38	26	1	0	-2.5	30.03	211.4	-41.4	0.0	0.0	0.6	0.4		X						Potentially Pathogenic	The methyl group of Ala399, located in an anti-parallel β sheet strand (res. Ala399-Ile411), is swapped for a hydroxyl-containing threonine. In the variant simulations, the hydroxyl group of Thr399 can form H-bonds with the backbone atoms of the residues in the membrane-facing loops (e.g., Gly382) in the C2 domain. Consequently, the ability of the Thr399 side chain to form H-bonds with the membrane-facing loops could adversely affect the dynamics and stability of the SynGAP-membrane association. However, since the effects on the dynamics of the membrane-facing loops can only be studied through the SynGAP-membrane complex, no definite conclusions can be drawn.
c.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.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.1286G>A	R429Q (3D Viewer)	Likely Benign	GAP	Uncertain		2	6-33438191-G-A	10	6.20e-6	-8.227	Likely Pathogenic	0.143	Likely Benign	Likely Benign	0.156	Likely Benign	0.45	Likely Benign	0.1	0.36	Likely Benign	0.41	Likely Benign	0.98	Ambiguous	-1.25	Neutral	1.000	Probably Damaging	0.979	Probably Damaging	3.47	Benign	0.58	Tolerated	3.38	25	1	1	1.0	-28.06	235.8	59.5	0.0	0.0	-0.3	0.4		X						Potentially Pathogenic	The guanidinium group of the Arg429 side chain, located in an α helix (res. Met414-Glu436), either forms a salt bridge with the carboxylate group of an acidic residue (Asp474, Asp467) or an H-bond with the hydroxyl group of Ser471 in an opposing α helix (res. Ala461-Phe476). In the variant simulations, Gln429 cannot form ionic interactions with the acidic residues; however, the carboxamide group can form multiple H-bonds. The H-bonding coordination of the Asn429 side chain varied between the replica simulations. In one simulation, three H-bonds were formed simultaneously with the Asp467 side chain, the backbone carbonyl group of Asn426, and the amide group of Met430 at the end of the same α helix. The residue swap could affect the tertiary structure assembly during folding due to weaker bond formation, but no large-scale negative effects were seen during the simulations.
c.1312G>A	A438T (3D Viewer)	Likely Benign	GAP	Conflicting		3	6-33438217-G-A	16	9.91e-6	-5.339	Likely Benign	0.085	Likely Benign	Likely Benign	0.021	Likely Benign	0.21	Likely Benign	0.0	-0.07	Likely Benign	0.07	Likely Benign	0.36	Likely Benign	-0.81	Neutral	0.300	Benign	0.011	Benign	4.18	Benign	0.14	Tolerated	3.38	26	1	0	-2.5	30.03	214.2	-42.7	-0.3	0.1	-0.4	0.1						X		Potentially Benign	The methyl group of Ala438, located in a four-residue loop connecting two α helices (res. Asn440-Thr458 and Pro413-Glu436), packs against hydrophobic residues from a nearby α helix or loop residues (e.g., Leu703, Val699). In the variant simulations, the methyl group of Thr438 is able to establish similar hydrophobic packing. Moreover, the hydroxyl group also H-bonds with nearby residues, such as the carbonyl group of the neighboring loop residue Pro437. Accordingly, the residue swap does not generate an apparent negative effect on the protein structure based on the simulations.
c.1622C>G	A541G (3D Viewer)		GAP	Uncertain		1	6-33438865-C-G	2	1.24e-6	-7.233	In-Between	0.341	Ambiguous	Likely Benign	0.421	Likely Benign	0.67	Ambiguous	0.0	0.94	Ambiguous	0.81	Ambiguous	0.76	Ambiguous	-1.48	Neutral	0.999	Probably Damaging	0.995	Probably Damaging	-1.31	Pathogenic	0.57	Tolerated	3.37	35	1	0	-2.2	-14.03	170.1	23.6	0.0	0.0	0.0	0.0	X							Potentially Pathogenic	Ala541 is located on the outer surface of an α-helix (res. Ala533-Val560). The methyl group of Ala541 is on the surface and does not form any interactions. Glycine, known as an “α-helix breaker,” weakens the integrity of the helix. Indeed, in the variant simulations, the hydrogen bond formation between Gly541 and the backbone carbonyl of Ala537 is disrupted.
c.1631G>A	R544Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33438874-G-A	1	6.20e-7	-10.281	Likely Pathogenic	0.596	Likely Pathogenic	Likely Benign	0.542	Likely Pathogenic	0.19	Likely Benign	0.2	0.87	Ambiguous	0.53	Ambiguous	1.40	Destabilizing	-2.41	Neutral	1.000	Probably Damaging	0.997	Probably Damaging	-1.40	Pathogenic	0.09	Tolerated	3.37	35	1	1	1.0	-28.06
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.163C>A	Q55K	Likely Benign		Uncertain		2	6-33423572-C-A	24	1.49e-5	-5.840	Likely Benign	0.612	Likely Pathogenic	Likely Benign	0.085	Likely Benign										-1.21	Neutral	0.140	Benign	0.184	Benign	3.91	Benign	0.00	Affected	4.32	1	1	1	-0.4	0.04
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.1424G>A	R475Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		2	6-33438456-G-A	5	3.10e-6	-12.087	Likely Pathogenic	0.721	Likely Pathogenic	Likely Benign	0.632	Likely Pathogenic	0.71	Ambiguous	0.1	0.12	Likely Benign	0.42	Likely Benign	0.82	Ambiguous	-3.65	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	-1.32	Pathogenic	0.01	Affected	3.39	28	1	1	1.0	-28.06	253.6	52.7	0.0	0.0	-0.8	0.0	X	X		X				Potentially Pathogenic	In the WT simulations, the guanidinium group of Arg475, located near the end of an α-helix (res. Ala461-Phe476), stacks with the phenyl ring of Phe476 and forms a salt bridge with Glu472. Additionally, Arg475 occasionally forms another salt bridge with the carboxylate group of Glu486 on the α-α loop connecting the two α-helices (res. Ala461-Phe476 and Leu489-Glu519) at the GAP-Ras interface. Therefore, Arg475 potentially plays a key role in positioning the loop by interacting with Glu486, which is necessary for the positioning of the “arginine finger” (Arg485) and, ultimately, for RasGTPase activation. In the variant simulations, Asn475 forms a hydrogen bond with Arg479 on the proceeding α-α loop. The absence of Phe476/Arg475 stacking and the Arg475-Glu472 salt bridge weakens the integrity of the terminal end of the α-helix during the variant simulations. Lastly, the potential effect of the residue swap on the SynGAP-Ras complex formation or GTPase activation cannot be fully addressed using the SynGAP solvent-only simulations.
c.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.1736G>A	R579Q (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440788-G-A	18	1.12e-5	-9.193	Likely Pathogenic	0.690	Likely Pathogenic	Likely Benign	0.673	Likely Pathogenic	0.65	Ambiguous	0.1	0.70	Ambiguous	0.68	Ambiguous	1.13	Destabilizing	-3.31	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.34	Pathogenic	0.06	Tolerated	3.37	34	1	1	1.0	-28.06
c.1738G>A	G580S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440790-G-A	1	6.20e-7	-10.788	Likely Pathogenic	0.861	Likely Pathogenic	Ambiguous	0.644	Likely Pathogenic	2.84	Destabilizing	0.2	0.59	Ambiguous	1.72	Ambiguous	0.87	Ambiguous	-5.73	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	-1.23	Pathogenic	0.07	Tolerated	3.37	34	1	0	-0.4	30.03	233.9	-49.3	0.8	0.0	0.6	0.1						X		Potentially Benign	Gly580 is located on the outer surface in a short α-α loop turn connecting two α-helices (res. Arg563-Glu578, res. Glu582-Phe608) in the WT simulations. In the variant simulations, the side chain of Ser580 faces outward, and its hydroxyl group does not make any new or additional interactions compared to Gly580 in the WT simulations that could affect the protein structure.
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.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.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.1918A>T	T640S (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441177-A-T	1	6.20e-7	-2.371	Likely Benign	0.062	Likely Benign	Likely Benign	0.088	Likely Benign	-0.78	Ambiguous	0.1	0.43	Likely Benign	-0.18	Likely Benign	-0.30	Likely Benign	0.92	Neutral	0.000	Benign	0.001	Benign	3.60	Benign	0.33	Tolerated	3.37	30	1	1	-0.1	-14.03
c.196C>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.196C>T	P66S	Likely Benign		Benign		1	6-33425804-C-T	2	1.24e-6	-2.760	Likely Benign	0.929	Likely Pathogenic	Ambiguous	0.081	Likely Benign										-1.69	Neutral	0.909	Possibly Damaging	0.641	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
c.1973G>A	G658D (3D Viewer)		GAP	Uncertain		1	6-33441232-G-A	3	1.86e-6	-7.786	In-Between	0.442	Ambiguous	Likely Benign	0.144	Likely Benign	-0.40	Likely Benign	0.1	-0.59	Ambiguous	-0.50	Ambiguous	0.46	Likely Benign	-2.64	Deleterious	0.008	Benign	0.005	Benign	3.53	Benign	0.38	Tolerated	3.39	24	1	-1	-3.1	58.04	219.8	-84.3	0.0	0.0	0.2	0.1						X		Potentially Pathogenic	Gly658, located on the outer surface of an α helix (res. Ser641-Glu666), weakens the helix integrity at that spot, which is necessary for the kink in the middle of the long helix. In the variant simulations, the carboxylic acid side chain of Asp658 is on the surface of the α helix and is not involved in any interactions. However, aspartate is not as effective a breaker of the secondary structure element as glycine, which may lead to misfolding.
c.1604G>C	S535T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33438847-G-C	14	8.67e-6	-3.886	Likely Benign	0.069	Likely Benign	Likely Benign	0.177	Likely Benign	0.45	Likely Benign	0.1	-0.27	Likely Benign	0.09	Likely Benign	0.17	Likely Benign	-0.81	Neutral	0.000	Benign	0.001	Benign	-1.25	Pathogenic	0.25	Tolerated	3.37	35	1	1	0.1	14.03	201.3	-17.3	-0.1	0.7	-0.2	0.1						X		Potentially Benign	Ser535 is located near the terminal end of an α-helix (res. Ala533-Val560) close to the membrane interface. In the WT simulations, the hydroxyl side chain of Ser535 forms hydrogen bonds with nearby residues (e.g., His539, Glu538) without any specific interactions. These hydrogen bonds disrupt the structure of the terminal end of the α-helix (Ala533-Ser535), causing it to weaken or unfold during the WT simulations. In the variant simulations, Thr535, a hydrophilic residue with a hydroxyl group of almost the same size as Ser, interacts more frequently with the preceding loop residues (e.g., Thr532, Cys531) due to its longer side chain. Regardless, the residue swap is tolerated in the simulations with no negative effects. However, due to its location near the SynGAP-membrane interface, the effect of the residue swap cannot be fully addressed using the SynGAP solvent-only simulations.	10.1016/j.ajhg.2020.11.011
c.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.1667A>G	N556S (3D Viewer)		GAP	Uncertain		1	6-33438910-A-G	3	1.86e-6	-6.576	Likely Benign	0.197	Likely Benign	Likely Benign	0.449	Likely Benign	0.52	Ambiguous	0.1	0.14	Likely Benign	0.33	Likely Benign	0.16	Likely Benign	-3.60	Deleterious	1.000	Probably Damaging	0.989	Probably Damaging	-1.22	Pathogenic	0.14	Tolerated	3.37	35	1	1	2.7	-27.03	198.8	31.0	0.0	0.0	-0.5	0.2						X		Potentially Benign	Asn556 is located on the outer surface of an α-helix (res. Ala533-Val560). The carboxamide group of Asn556 forms hydrogen bonds with nearby residues such as Lys553 and Cys552. It also forms a hydrogen bond with the backbone carbonyl group of Cys552, which weakens the α-helix integrity. In the variant simulations, the hydroxyl group of Ser556 forms a more stable hydrogen bond with the backbone carbonyl oxygen of the same helix residue, Cys552, compared to Asn556 in the WT. Serine has a slightly lower propensity to reside in an α-helix than asparagine, which may exacerbate the negative effect on the α-helix integrity. However, the residue swap does not cause negative structural effects during the simulations.
c.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.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.2111G>C	S704T (3D Viewer)	Likely Benign	GAP	Uncertain		1				-4.930	Likely Benign	0.265	Likely Benign	Likely Benign	0.071	Likely Benign	0.80	Ambiguous	0.0	0.15	Likely Benign	0.48	Likely Benign	0.29	Likely Benign	-1.72	Neutral	0.525	Possibly Damaging	0.107	Benign	3.45	Benign	0.07	Tolerated	3.47	10	1	1	0.1	14.03	201.7	-18.0	0.0	0.0	-0.2	0.7						X		Potentially Benign	Ser704 is located at the end and outer surface of an α-helix (res. Thr704-Gly712), which is connected via a tight turn or loop to another α-helix (res. Asp684-Gln702). The hydroxyl side chain of Ser704 occasionally forms a hydrogen bond with the amide group of Ala707. Similarly, in the variant simulations, the hydroxyl side chain of Thr704 forms hydrogen bonds with the amide groups of Ala707 and Leu708. Thus, the residue swap does not cause any apparent structural change.
c.2113A>C	K705Q (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441372-A-C	1	6.20e-7	-5.787	Likely Benign	0.436	Ambiguous	Likely Benign	0.142	Likely Benign	-0.10	Likely Benign	0.0	0.33	Likely Benign	0.12	Likely Benign	-0.02	Likely Benign	-0.24	Neutral	0.997	Probably Damaging	0.969	Probably Damaging	3.42	Benign	0.78	Tolerated	3.47	10	1	1	0.4	-0.04
c.2115G>C	K705N (3D Viewer)	Likely Pathogenic	GAP	Likely Pathogenic		1				-9.767	Likely Pathogenic	0.925	Likely Pathogenic	Ambiguous	0.183	Likely Benign	0.74	Ambiguous	0.0	0.37	Likely Benign	0.56	Ambiguous	0.44	Likely Benign	-3.12	Deleterious	0.996	Probably Damaging	0.876	Possibly Damaging	3.37	Benign	0.02	Affected	3.47	10	1	0	0.4	-14.07	221.4	-20.2	0.0	0.0	0.0	0.1						X		Uncertain	The amino side chain of Lys705, located at the end and outer surface of an α-helix (res. Thr704-Gly712), does not form any interactions in the WT simulations. In the variant simulations, the carboxamide side chain of Asn705 briefly forms a salt bridge with Glu706. However, there is no apparent difference between the systems. Due to the model ending abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2131C>G	L711V (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33441596-C-G	1	6.20e-7	-10.045	Likely Pathogenic	0.709	Likely Pathogenic	Likely Benign	0.170	Likely Benign	3.48	Destabilizing	0.1	2.22	Destabilizing	2.85	Destabilizing	1.40	Destabilizing	-2.59	Deleterious	0.992	Probably Damaging	0.970	Probably Damaging	3.34	Benign	0.00	Affected	3.50	9	1	2	0.4	-14.03
c.2158G>A	D720N (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441623-G-A	5	3.10e-6	-9.135	Likely Pathogenic	0.654	Likely Pathogenic	Likely Benign	0.289	Likely Benign	0.01	Likely Benign	0.0	-0.20	Likely Benign	-0.10	Likely Benign	0.46	Likely Benign	-3.74	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	2.18	Pathogenic	0.01	Affected	3.50	9	1	2	0.0	-0.98
c.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.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.2200C>T	P734S	Likely Benign		Uncertain		2	6-33441665-C-T	2	1.24e-6	-4.291	Likely Benign	0.077	Likely Benign	Likely Benign	0.030	Likely Benign										-2.44	Neutral	0.344	Benign	0.048	Benign	2.77	Benign	0.11	Tolerated	3.64	6	1	-1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.1729G>A	A577T (3D Viewer)	Likely Benign	GAP	Benign		1	6-33440781-G-A	6	3.72e-6	-5.311	Likely Benign	0.322	Likely Benign	Likely Benign	0.427	Likely Benign	0.86	Ambiguous	0.1	0.54	Ambiguous	0.70	Ambiguous	0.54	Ambiguous	-1.47	Neutral	0.999	Probably Damaging	0.987	Probably Damaging	-1.31	Pathogenic	0.47	Tolerated	3.37	34	1	0	-2.5	30.03	191.9	-43.4	0.0	0.0	0.7	0.1						X		Potentially Benign	Ala577 is located near the end and outer surface of an α-helix (res. Arg563-Glu578), where its methyl group does not form any particular interactions in the WT simulations. In the variant simulations, the hydroxyl group of the Thr577 side chain hydrogen bonds with the backbone atoms of Arg573 and Lys574 within the same helix, which has the potential to weaken the stability of the secondary structure element. Regardless, the residue swap seems to be well tolerated based on the variant simulations.
c.1730C>G	A577G (3D Viewer)	Likely Benign	GAP	Benign/Likely benign		2	6-33440782-C-G	1	6.20e-7	-5.717	Likely Benign	0.268	Likely Benign	Likely Benign	0.443	Likely Benign	0.83	Ambiguous	0.0	1.02	Ambiguous	0.93	Ambiguous	0.86	Ambiguous	-1.84	Neutral	0.997	Probably Damaging	0.990	Probably Damaging	-1.31	Pathogenic	0.31	Tolerated	3.37	34	1	0	-2.2	-14.03	158.7	23.6	0.0	0.0	0.0	0.0						X		Potentially Benign	Ala577 is located near the end and outer surface of an α-helix (res. Arg563-Glu578), where its methyl group does not form any particular interactions in the WT simulations. The introduced residue, glycine, is known as an “α-helix breaker.” However, the residue swap caused only minor helix shortening in one of the replica simulations for the variant system. Regardless, the residue swap seems to be well tolerated based on the variant simulations.
c.2219G>A	R740Q	Likely Benign		Uncertain		1	6-33441684-G-A	4	2.48e-6	-5.195	Likely Benign	0.078	Likely Benign	Likely Benign	0.102	Likely Benign										-0.67	Neutral	0.999	Probably Damaging	0.881	Possibly Damaging	2.60	Benign	0.08	Tolerated	4.32	2	1	1	1.0	-28.06
c.221G>A	S74N	Likely Benign		Uncertain		1	6-33425829-G-A	5	3.10e-6	-5.156	Likely Benign	0.112	Likely Benign	Likely Benign	0.031	Likely Benign										-0.89	Neutral	0.043	Benign	0.007	Benign	4.09	Benign	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.2221C>T	P741S	Likely Benign		Uncertain		2	6-33441686-C-T	3	1.86e-6	-3.700	Likely Benign	0.063	Likely Benign	Likely Benign	0.076	Likely Benign										-0.27	Neutral	0.270	Benign	0.136	Benign	2.92	Benign	0.00	Affected	4.32	2	1	-1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.2225G>A	R742Q	Likely Benign		Uncertain		2	6-33441690-G-A	24	1.49e-5	-4.090	Likely Benign	0.068	Likely Benign	Likely Benign	0.054	Likely Benign										-0.19	Neutral	0.032	Benign	0.007	Benign	2.73	Benign	0.07	Tolerated	4.32	2	1	1	1.0	-28.06
c.1742G>A	R581Q (3D Viewer)	Likely Pathogenic	GAP	Benign		1	6-33440794-G-A	8	4.96e-6	-7.584	In-Between	0.673	Likely Pathogenic	Likely Benign	0.481	Likely Benign	1.31	Ambiguous	0.1	-0.42	Likely Benign	0.45	Likely Benign	0.88	Ambiguous	-2.77	Deleterious	1.000	Probably Damaging	0.995	Probably Damaging	-1.21	Pathogenic	0.11	Tolerated	3.37	34	1	1	1.0	-28.06	239.6	53.5	-0.2	0.2	-0.4	0.1		X						Potentially Pathogenic	Arg581 is located on a short α-α loop between two α helices (res. Arg563-Glu578 and res. Glu582-Ser604). In the WT simulations, the guanidinium group of Arg581 forms salt bridges with the carboxylate groups of Asp583 within the same helix, as well as with Glu478 and/or Glu480 on a slightly α-helical loop (res. Glu478-Thr488) preceding another α helix (res. Ala461-Phe476).In the variant simulations, the neutral carboxamide group of the Gln581 side chain cannot form any of these salt bridges. Instead, it packs hydrophobically against Met477 and Ile587 or forms hydrogen bonds sporadically with nearby residues (e.g., Asp583, Arg587). Thus, although no drastic changes are observed in the variant simulations, the residue swap could weaken the tertiary structure assembly.
c.2246G>A	R749Q	Likely Benign		Likely Benign		1	6-33441711-G-A	4	2.48e-6	-3.069	Likely Benign	0.212	Likely Benign	Likely Benign	0.152	Likely Benign										-1.00	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	2.64	Benign	0.03	Affected	4.32	2	1	1	1.0	-28.06
c.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.1768A>G	S590G (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2	6-33440820-A-G	14	8.67e-6	-14.277	Likely Pathogenic	0.574	Likely Pathogenic	Likely Benign	0.379	Likely Benign	0.67	Ambiguous	0.1	1.28	Ambiguous	0.98	Ambiguous	0.71	Ambiguous	-3.92	Deleterious	1.000	Probably Damaging	0.922	Probably Damaging	3.42	Benign	0.06	Tolerated	3.37	35	1	0	0.4	-30.03	186.7	49.4	0.0	0.0	0.1	0.0		X						Potentially Pathogenic	In the WT simulations, the hydroxyl group of Ser590, located on an α helix (res. Glu582-Met603), forms hydrogen bonds with the backbone carbonyl of Ala634 and/or the carboxamide group of the Asn635 side chain at the end of the opposing α helix (res. Thr619-Ala634).The residue swap could weaken the integrity of the α helix, as glycine is known as an “α helix breaker.” However, no discernible difference was observed between the WT and variant simulations in this regard. Importantly, Gly590 cannot form hydrogen bonds with the opposing helix in the same way that serine can, which could weaken the tertiary structure assembly between the two helices.
c.1771G>A	A591T (3D Viewer)	Likely Pathogenic	GAP	Conflicting		3	6-33440823-G-A	18	1.12e-5	-9.572	Likely Pathogenic	0.704	Likely Pathogenic	Likely Benign	0.270	Likely Benign	1.61	Ambiguous	0.2	1.00	Ambiguous	1.31	Ambiguous	1.19	Destabilizing	-3.40	Deleterious	0.955	Possibly Damaging	0.209	Benign	3.48	Benign	0.01	Affected	3.37	35	1	0	-2.5	30.03	202.9	-43.4	0.2	0.0	0.7	0.1						X		Potentially Benign	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, the hydroxyl group of Thr591 can form hydrogen bonds with the backbone carbonyl of Ile843 in the opposing loop or the backbone carbonyl group of Arg587. These interactions could either reinforce the tertiary assembly or weaken the α helix unity. Additionally, the Thr591 side chain can hydrogen bond with the guanidinium group of the Arg587 side chain, potentially strengthening the α helix unity.Overall, the residue swap does not seem to cause any major negative effects on the protein structure.
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.2277G>A	M759I	Likely Benign		Uncertain		1	6-33441742-G-A	1	6.20e-7	-4.058	Likely Benign	0.393	Ambiguous	Likely Benign	0.075	Likely Benign										-0.88	Neutral	0.454	Possibly Damaging	0.192	Benign	2.83	Benign	0.34	Tolerated	3.99	5	1	2	2.6	-18.03
c.2282G>A	R761Q	Likely Benign		Uncertain		1	6-33441747-G-A	11	6.81e-6	-4.187	Likely Benign	0.202	Likely Benign	Likely Benign	0.191	Likely Benign										-0.63	Neutral	0.996	Probably Damaging	0.878	Possibly Damaging	2.75	Benign	0.40	Tolerated	3.99	5	1	1	1.0	-28.06
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.2302G>A	D768N	Likely Benign		Uncertain		1	6-33442460-G-A	2	2.57e-6	-6.892	Likely Benign	0.453	Ambiguous	Likely Benign	0.048	Likely Benign										-0.77	Neutral	0.106	Benign	0.009	Benign	4.07	Benign	0.96	Tolerated	3.64	6	1	2	0.0	-0.98
c.2324G>A	R775Q	Likely Benign		Conflicting		3	6-33442482-G-A	11	1.41e-5	-4.476	Likely Benign	0.229	Likely Benign	Likely Benign	0.085	Likely Benign										-0.63	Neutral	0.969	Probably Damaging	0.863	Possibly Damaging	4.17	Benign	0.16	Tolerated	3.64	6	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.2343G>A	M781I	Likely Benign		Benign		1				-2.484	Likely Benign	0.323	Likely Benign	Likely Benign	0.101	Likely Benign										0.05	Neutral	0.000	Benign	0.001	Benign	2.89	Benign	1.00	Tolerated	3.64	6	1	2	2.6	-18.03
c.2349G>A	M783I	Likely Benign		Benign		1	6-33442901-G-A	6	3.72e-6	-3.560	Likely Benign	0.418	Ambiguous	Likely Benign	0.042	Likely Benign										-0.54	Neutral	0.004	Benign	0.006	Benign	2.87	Benign	0.22	Tolerated	3.64	6	1	2	2.6	-18.03
c.2350G>A	A784T	Likely Benign		Benign		1				-3.579	Likely Benign	0.089	Likely Benign	Likely Benign	0.046	Likely Benign										1.23	Neutral	0.001	Benign	0.006	Benign	2.92	Benign	1.00	Tolerated	3.64	6	1	0	-2.5	30.03
c.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.2362T>A	S788T	Likely Benign	SH3-binding motif	Uncertain		2	6-33442914-T-A	4	2.49e-6	-4.288	Likely Benign	0.288	Likely Benign	Likely Benign	0.092	Likely Benign										-2.25	Neutral	0.979	Probably Damaging	0.982	Probably Damaging	1.55	Pathogenic	0.02	Affected	3.64	6	1	1	0.1	14.03
c.2369C>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.1862G>A	R621Q (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33440914-G-A	19	1.18e-5	-14.682	Likely Pathogenic	0.910	Likely Pathogenic	Ambiguous	0.621	Likely Pathogenic	0.81	Ambiguous	0.1	1.13	Ambiguous	0.97	Ambiguous	1.35	Destabilizing	-3.98	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	2.82	Benign	0.01	Affected	3.37	35	1	1	1.0	-28.06	243.7	54.3	0.0	0.0	-0.4	0.2		X		X				Potentially Pathogenic	The guanidinium group of Arg621, located in an α helix (res. Glu617-Asn635), forms a salt bridge with Glu525 in a nearby loop and stacks with Leu635. In the variant simulations, the carboxamide side chain of Gln621, which can act as both a hydrogen bond acceptor and donor, also stacks with Leu635 but can only sporadically hydrogen bond with Glu525.Accordingly, the residue swap could affect the tertiary structure integrity by disrupting the salt bridge formation. Additionally, due to its location at the GAP-Ras interface, the residue swap could impact the complex formation with the GTPase, but this cannot be investigated using solvent-only simulations.
c.1904A>G	N635S (3D Viewer)		GAP	Conflicting		4	6-33440956-A-G	10	6.20e-6	-9.002	Likely Pathogenic	0.101	Likely Benign	Likely Benign	0.104	Likely Benign	0.80	Ambiguous	0.1	0.67	Ambiguous	0.74	Ambiguous	0.95	Ambiguous	-4.45	Deleterious	0.261	Benign	0.044	Benign	3.06	Benign	0.05	Affected	3.37	34	1	1	2.7	-27.03	196.0	30.9	0.1	0.0	-0.3	0.2				X				Uncertain	In the WT simulations, the carboxamide side chain of Asn635, located on the outer surface of an α helix (res. Glu617-Asn635), forms hydrogen bonds with Gln631 on the same α helix and with the hydroxyl side chain of Ser590 on an opposing α helix (res. Glu582-Met603).In the variant simulations, the side chain of Ser635 is shorter than asparagine and thus prefers to hydrogen bond with the carbonyl group of Gln631 on the same helix and, to a lesser extent, with Ser590 compared to Asn635 in the WT. Ser635 forms hydrogen bonds with the backbone atoms of the same helix, which may destabilize the helix, although this is not clearly evident in the simulations. The weakening of the hydrogen bond between Ser635 and Ser590 in the variant may also weaken the tertiary structure assembly between the helices.Additionally, Asn635 is at the GTPase interface. However, the implication of the residue swap on the complex formation with the GTPase cannot be investigated using solvent-only simulations.
c.2405G>A	G802D	Likely Benign	SH3-binding motif	Uncertain		1	6-33442957-G-A	1	6.20e-7	-5.083	Likely Benign	0.476	Ambiguous	Likely Benign	0.153	Likely Benign										-0.38	Neutral	0.126	Benign	0.138	Benign	2.72	Benign	0.09	Tolerated	3.77	5	1	-1	-3.1	58.04
c.2434C>T	P812S	Likely Benign	SH3-binding motif	Uncertain		1	6-33442986-C-T	1	6.20e-7	-5.689	Likely Benign	0.456	Ambiguous	Likely Benign	0.162	Likely Benign										-0.62	Neutral	0.999	Probably Damaging	0.966	Probably Damaging	2.89	Benign	0.95	Tolerated	4.32	4	1	-1	0.8	-10.04
c.2014A>G	T672A (3D Viewer)	Likely Benign	GAP	Benign		1	6-33441273-A-G	3	1.86e-6	-6.524	Likely Benign	0.109	Likely Benign	Likely Benign	0.046	Likely Benign	0.51	Ambiguous	0.3	1.15	Ambiguous	0.83	Ambiguous	0.65	Ambiguous	-3.20	Deleterious	0.006	Benign	0.002	Benign	3.44	Benign	0.12	Tolerated	3.40	25	1	0	2.5	-30.03	188.5	42.5	-0.1	0.3	0.2	0.0		X						Potentially Pathogenic	The hydroxyl group of Thr672, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop itself, such as Lys566, Glu666, and Asn669. In the variant simulations, Ala672 can only form a hydrogen bond with Lys566 via its backbone carbonyl group. Consequently, it cannot maintain the Lys566-Glu666 salt bridge through hydrogen bonding, leading to a significant disruption of the intricate and stable hydrogen-bond network between the loop and the helices.
c.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.2506A>G	S836G	Likely Benign		Uncertain		1	6-33443058-A-G	4	2.48e-6	-4.749	Likely Benign	0.112	Likely Benign	Likely Benign	0.066	Likely Benign										-1.65	Neutral	0.006	Benign	0.019	Benign	2.54	Benign	0.39	Tolerated	3.77	5	1	0	0.4	-30.03
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.2521G>A	V841M			Uncertain		1	6-33443073-G-A	3	1.86e-6	-7.000	In-Between	0.651	Likely Pathogenic	Likely Benign	0.119	Likely Benign										-0.74	Neutral	0.999	Probably Damaging	0.998	Probably Damaging	2.54	Benign	0.02	Affected	3.77	5	1	2	-2.3	32.06
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.2567A>G	N856S	Likely Benign		Uncertain		1	6-33443119-A-G	2	1.24e-6	-2.104	Likely Benign	0.064	Likely Benign	Likely Benign	0.040	Likely Benign										-1.54	Neutral	0.901	Possibly Damaging	0.535	Possibly Damaging	4.16	Benign	0.30	Tolerated	3.88	3	1	1	2.7	-27.03
c.2573G>A	S858N	Likely Benign		Uncertain		1	6-33443125-G-A	2	1.24e-6	-4.311	Likely Benign	0.121	Likely Benign	Likely Benign	0.107	Likely Benign										-0.67	Neutral	0.448	Benign	0.846	Possibly Damaging	4.13	Benign	0.02	Affected	3.77	5	1	1	-2.7	27.03
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.2111G>A	S704N (3D Viewer)	Likely Benign	GAP	Benign/Likely benign		3	6-33441370-G-A	27	1.67e-5	-5.917	Likely Benign	0.421	Ambiguous	Likely Benign	0.058	Likely Benign	0.48	Likely Benign	0.1	-0.12	Likely Benign	0.18	Likely Benign	0.54	Ambiguous	-0.49	Neutral	0.771	Possibly Damaging	0.275	Benign	3.39	Benign	0.08	Tolerated	3.47	10	1	1	-2.7	27.03	233.2	-29.1	-0.1	0.0	-0.1	0.1						X		Potentially Benign	Ser704 is located at the end and outer surface of an α-helix (res. Thr704-Gly712), which is connected via a tight turn or loop to another α-helix (res. Asp684-Gln702). The hydroxyl side chain of Ser704 occasionally forms a hydrogen bond with the amide group of Ala707. However, in the variant simulations, the carboxamide side chain of Asn704 achieves more lasting and numerous hydrogen-bonding interactions with the residues at the helix end, such as Glu706, Ala707, and Leu708. Consequently, the residue swap could strengthen the α-helix secondary structure integrity at the helix end, which could have either positive or negative effects on its function.
c.2143C>T	P715S (3D Viewer)		GAP	Likely Pathogenic		1	6-33441608-C-T	1	6.20e-7	-7.635	In-Between	0.787	Likely Pathogenic	Ambiguous	0.277	Likely Benign	3.54	Destabilizing	0.0	0.81	Ambiguous	2.18	Destabilizing	0.94	Ambiguous	-7.17	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.43	Benign	0.01	Affected	3.50	9	1	-1	0.8	-10.04	231.8	-14.0	-0.1	0.0	-0.8	0.1							X	Uncertain	Pro715, along with Gly712 and Pro713, are located in a hinge region of an α-helix making a ~90-degree turn (res. Lys705-Leu725). In the WT simulations, the pyrrolidine side chain of Pro715, lacking the backbone amide groups altogether, forces the tight helix turn to take place while also hydrophobically packing with nearby residues (e.g., Leu700, Leu708, Leu714, and Leu718). Leu715, with a normal amide backbone, could potentially affect protein folding and turn formation, although this was not observed in the variant simulations. Additionally, the hydroxyl group of the Ser715 side chain can form hydrogen bonds with the backbone carbonyl group of Gly712 and disrupt the hydrophobic packing arrangement of the leucine residues from the neighboring α-helices, impacting the GAP domain tertiary assembly.
c.2147G>A	R716Q (3D Viewer)		GAP	Conflicting		2	6-33441612-G-A	4	2.48e-6	-8.338	Likely Pathogenic	0.308	Likely Benign	Likely Benign	0.210	Likely Benign	-0.01	Likely Benign	0.0	0.47	Likely Benign	0.23	Likely Benign	0.58	Ambiguous	-3.14	Deleterious	1.000	Probably Damaging	0.990	Probably Damaging	3.35	Benign	0.02	Affected	3.50	9	1	1	1.0	-28.06	250.0	48.9	0.0	0.0	-0.5	0.0						X		Uncertain	The guanidinium group of Arg716, located on the outer surface of an α-helix (res. Leu714-Arg726), forms a salt bridge with the carboxylate group of Asp720. In the variant simulations, the carboxamide group of Gln716 also forms a hydrogen bond with the carboxylate group of Asp720, although this bond is weaker than the Arg716 salt bridge in the WT. Overall, no adverse effects on the protein structure are observed in the simulations. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.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
c.2651G>A	R884Q	Likely Benign		Uncertain		2	6-33443203-G-A	5	3.10e-6	-3.785	Likely Benign	0.128	Likely Benign	Likely Benign	0.055	Likely Benign										-0.42	Neutral	0.012	Benign	0.004	Benign	2.62	Benign	0.36	Tolerated	4.32	4	1	1	1.0	-28.06
c.265C>G	P89A	Likely Benign		Uncertain		2				-5.778	Likely Benign	0.920	Likely Pathogenic	Ambiguous	0.095	Likely Benign										-2.47	Neutral	0.225	Benign	0.020	Benign	3.77	Benign	0.00	Affected	4.32	1	1	-1	3.4	-26.04
c.2684G>A	S895N	Likely Benign		Uncertain		1				-6.399	Likely Benign	0.604	Likely Pathogenic	Likely Benign	0.118	Likely Benign										-0.85	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.64	Benign	0.30	Tolerated	4.32	4	1	1	-2.7	27.03
c.2704G>A	A902T	Likely Benign		Likely Benign		1	6-33443256-G-A	36	2.23e-5	-4.966	Likely Benign	0.116	Likely Benign	Likely Benign	0.075	Likely Benign										-1.11	Neutral	0.951	Possibly Damaging	0.617	Possibly Damaging	2.61	Benign	0.01	Affected	3.77	5	1	0	-2.5	30.03
c.2729G>C	G910A	Likely Benign		Uncertain		1	6-33443281-G-C	1	6.20e-7	-3.587	Likely Benign	0.361	Ambiguous	Likely Benign	0.209	Likely Benign										-1.43	Neutral	0.999	Probably Damaging	0.999	Probably Damaging	2.78	Benign	0.10	Tolerated	3.77	5	1	0	2.2	14.03
c.2743G>A	G915S	Likely Benign		Benign		1	6-33443295-G-A	9	5.58e-6	-3.557	Likely Benign	0.083	Likely Benign	Likely Benign	0.050	Likely Benign										-0.88	Neutral	0.801	Possibly Damaging	0.201	Benign	2.73	Benign	0.31	Tolerated	3.77	5	1	0	-0.4	30.03
c.2765G>A	R922Q	Likely Benign		Benign		1	6-33443317-G-A	7	4.34e-6	-3.295	Likely Benign	0.189	Likely Benign	Likely Benign	0.085	Likely Benign										-0.27	Neutral	0.992	Probably Damaging	0.736	Possibly Damaging	2.57	Benign	0.20	Tolerated	3.77	5	1	1	1.0	-28.06
c.280C>T	P94S	Likely Benign		Benign		1	6-33425888-C-T	5	3.10e-6	-3.151	Likely Benign	0.084	Likely Benign	Likely Benign	0.093	Likely Benign										-2.36	Neutral	0.092	Benign	0.008	Benign	4.13	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
c.2818G>A	G940S	Likely Benign		Uncertain		1	6-33443370-G-A	1	6.20e-7	-5.451	Likely Benign	0.084	Likely Benign	Likely Benign	0.135	Likely Benign										0.45	Neutral	0.409	Benign	0.253	Benign	2.77	Benign	0.44	Tolerated	3.77	5	1	0	-0.4	30.03
c.2830G>A	G944S	Likely Benign		Benign		1	6-33443382-G-A	13	8.05e-6	-5.303	Likely Benign	0.082	Likely Benign	Likely Benign	0.223	Likely Benign										-0.75	Neutral	0.007	Benign	0.004	Benign	3.77	Benign	0.00	Affected	4.32	4	1	0	-0.4	30.03
c.2840G>C	G947A	Likely Benign		Likely Benign		1	6-33443392-G-C	28	1.73e-5	-6.511	Likely Benign	0.080	Likely Benign	Likely Benign	0.156	Likely Benign										-0.41	Neutral	0.224	Benign	0.131	Benign	4.97	Benign	0.10	Tolerated	4.32	4	1	0	2.2	14.03
c.2845G>A	G949S	Likely Benign		Benign/Likely benign		4	6-33443397-G-A	122	7.56e-5	-5.693	Likely Benign	0.072	Likely Benign	Likely Benign	0.321	Likely Benign										0.30	Neutral	0.611	Possibly Damaging	0.102	Benign	2.23	Pathogenic	0.00	Affected	4.32	4	1	0	-0.4	30.03																10.1016/j.ajhg.2020.11.011
c.2854G>A	G952S	Likely Benign		Conflicting		2	6-33443406-G-A	2	1.24e-6	-6.190	Likely Benign	0.077	Likely Benign	Likely Benign	0.167	Likely Benign										0.19	Neutral	0.000	Benign	0.002	Benign	3.31	Benign	0.07	Tolerated	3.77	5	1	0	-0.4	30.03
c.2860C>T	P954S	Likely Benign		Likely Benign		1	6-33443412-C-T	1	6.20e-7	-3.525	Likely Benign	0.062	Likely Benign	Likely Benign	0.143	Likely Benign										-0.25	Neutral	0.954	Possibly Damaging	0.812	Possibly Damaging	2.87	Benign	1.00	Tolerated	3.77	5	1	-1	0.8	-10.04
c.2863T>C	S955P	Likely Benign		Uncertain		1	6-33443415-T-C	3	1.86e-6	-2.584	Likely Benign	0.073	Likely Benign	Likely Benign	0.098	Likely Benign										-0.75	Neutral	0.001	Benign	0.004	Benign	2.33	Pathogenic	0.00	Affected	3.77	5	1	-1	-0.8	10.04
c.286G>A	G96S	Likely Benign		Uncertain		1	6-33425894-G-A	5	3.10e-6	-3.049	Likely Benign	0.065	Likely Benign	Likely Benign	0.071	Likely Benign										-0.76	Neutral	0.364	Benign	0.008	Benign	4.25	Benign	0.00	Affected	4.32	1	1	0	-0.4	30.03
c.2900G>A	R967Q	Likely Benign		Benign/Likely benign		2	6-33443452-G-A	31	1.92e-5	-3.057	Likely Benign	0.080	Likely Benign	Likely Benign	0.104	Likely Benign										-0.01	Neutral	0.994	Probably Damaging	0.626	Possibly Damaging	4.21	Benign	0.36	Tolerated	4.32	2	1	1	1.0	-28.06
c.2924C>G	T975S	Likely Benign		Uncertain		1				-2.743	Likely Benign	0.068	Likely Benign	Likely Benign	0.109	Likely Benign										-0.57	Neutral	0.059	Benign	0.061	Benign	4.16	Benign	0.20	Tolerated			1	1	-0.1	-14.03
c.2932C>T	P978S	Likely Benign		Uncertain		1				-3.913	Likely Benign	0.151	Likely Benign	Likely Benign	0.085	Likely Benign										-1.07	Neutral	0.481	Possibly Damaging	0.220	Benign	4.22	Benign	0.48	Tolerated			1	-1	0.8	-10.04
c.2948G>A	S983N			Likely Benign		1	6-33443500-G-A	6	3.72e-6	-5.604	Likely Benign	0.909	Likely Pathogenic	Ambiguous	0.136	Likely Benign										-1.78	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.04	Pathogenic	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.2954G>A	S985N	Likely Benign		Uncertain		1				-6.979	Likely Benign	0.845	Likely Pathogenic	Ambiguous	0.088	Likely Benign										-1.68	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.65	Benign	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.2960A>G	D987G	Likely Pathogenic		Uncertain		1				-4.782	Likely Benign	0.849	Likely Pathogenic	Ambiguous	0.234	Likely Benign										-2.79	Deleterious	0.943	Possibly Damaging	0.808	Possibly Damaging	2.45	Pathogenic	0.07	Tolerated	4.32	2	1	-1	3.1	-58.04
c.2983C>T	P995S	Likely Benign		Uncertain		1				-4.457	Likely Benign	0.071	Likely Benign	Likely Benign	0.042	Likely Benign										-1.03	Neutral	0.011	Benign	0.015	Benign	4.24	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
c.2989G>A	A997T	Likely Benign		Uncertain		1				-4.102	Likely Benign	0.071	Likely Benign	Likely Benign	0.085	Likely Benign										-0.62	Neutral	0.224	Benign	0.120	Benign	4.17	Benign	0.00	Affected	4.32	4	1	0	-2.5	30.03
c.29G>A	R10Q	Likely Benign		Uncertain		2	6-33420293-G-A	20	1.30e-5	-4.438	Likely Benign	0.185	Likely Benign	Likely Benign	0.084	Likely Benign										0.03	Neutral	0.121	Benign	0.004	Benign	4.17	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06
c.3020G>A	S1007N	Likely Benign		Benign		1				-5.113	Likely Benign	0.803	Likely Pathogenic	Ambiguous	0.075	Likely Benign										-1.54	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.65	Benign	0.01	Affected	3.77	5	1	1	-2.7	27.03
c.3100C>G	P1034A	Likely Benign		Benign		1				-4.174	Likely Benign	0.178	Likely Benign	Likely Benign	0.060	Likely Benign										-2.44	Neutral	0.059	Benign	0.061	Benign	2.47	Pathogenic	0.06	Tolerated	3.77	5	1	-1	3.4	-26.04
c.3121C>T	P1041S	Likely Benign		Conflicting		2	6-33443673-C-T	1	6.20e-7	-4.246	Likely Benign	0.121	Likely Benign	Likely Benign	0.344	Likely Benign										-2.72	Deleterious	0.664	Possibly Damaging	0.283	Benign	5.48	Benign	0.11	Tolerated	3.77	5	1	-1	0.8	-10.04
c.3125A>G	Q1042R	Likely Benign		Uncertain		2	6-33443677-A-G	2	1.24e-6	-2.928	Likely Benign	0.413	Ambiguous	Likely Benign	0.300	Likely Benign										-1.39	Neutral	0.586	Possibly Damaging	0.120	Benign	5.48	Benign	0.12	Tolerated	3.77	5	1	1	-1.0	28.06
c.3134C>G	A1045G	Likely Benign		Benign/Likely benign		7	6-33443686-C-G	1407	8.72e-4	-3.246	Likely Benign	0.075	Likely Benign	Likely Benign	0.024	Likely Benign										-1.21	Neutral	0.224	Benign	0.066	Benign	2.64	Benign	0.33	Tolerated	3.77	5	1	0	-2.2	-14.03																10.1016/j.ajhg.2020.11.011
c.3160G>A	G1054S	Likely Benign		Benign		1	6-33443712-G-A	32	1.99e-5	-5.294	Likely Benign	0.075	Likely Benign	Likely Benign	0.160	Likely Benign										0.21	Neutral	0.121	Benign	0.013	Benign	4.04	Benign	0.63	Tolerated	3.77	5	1	0	-0.4	30.03
c.3161G>A	G1054D			Uncertain		1				-10.385	Likely Pathogenic	0.351	Ambiguous	Likely Benign	0.279	Likely Benign										-0.26	Neutral	0.818	Possibly Damaging	0.266	Benign	4.07	Benign	0.37	Tolerated	3.77	5	1	-1	-3.1	58.04
c.3170G>A	S1057N	Likely Benign		Uncertain		1				-6.386	Likely Benign	0.117	Likely Benign	Likely Benign	0.218	Likely Benign										-0.41	Neutral	0.451	Benign	0.129	Benign	5.25	Benign	0.28	Tolerated			1	1	-2.7	27.03
c.3172G>A	G1058S	Likely Benign		Conflicting		3	6-33443724-G-A	114	7.08e-5	-5.178	Likely Benign	0.081	Likely Benign	Likely Benign	0.108	Likely Benign										0.26	Neutral	0.001	Benign	0.001	Benign	5.38	Benign	0.04	Affected	3.77	5	1	0	-0.4	30.03
c.3176G>C	G1059A	Likely Benign		Uncertain		1	6-33443728-G-C	4	2.49e-6	-6.754	Likely Benign	0.081	Likely Benign	Likely Benign	0.329	Likely Benign										-0.17	Neutral	0.001	Benign	0.002	Benign	2.56	Benign	0.00	Affected	4.32	2	1	0	2.2	14.03
c.3178G>A	G1060S	Likely Benign		Uncertain		1	6-33443730-G-A			-4.759	Likely Benign	0.082	Likely Benign	Likely Benign	0.376	Likely Benign										-0.08	Neutral	0.271	Benign	0.054	Benign	2.69	Benign	0.49	Tolerated	4.32	2	1	0	-0.4	30.03
c.3181G>A	G1061S	Likely Benign		Uncertain		1				-4.891	Likely Benign	0.079	Likely Benign	Likely Benign	0.283	Likely Benign										-0.68	Neutral	0.004	Benign	0.004	Benign	4.00	Benign	0.00	Affected			1	0	-0.4	30.03
c.3196C>T	P1066S	Likely Benign		Likely Pathogenic		1				-4.746	Likely Benign	0.070	Likely Benign	Likely Benign	0.145	Likely Benign										-2.47	Neutral	0.972	Probably Damaging	0.850	Possibly Damaging	2.74	Benign	0.00	Affected	4.32	2	1	-1	0.8	-10.04
c.3223C>A	Q1075K	Likely Benign		Uncertain		1				-5.135	Likely Benign	0.728	Likely Pathogenic	Likely Benign	0.134	Likely Benign										-0.67	Neutral	0.963	Probably Damaging	0.959	Probably Damaging	2.75	Benign	1.00	Tolerated	3.77	5	1	1	-0.4	0.04
c.3238G>A	A1080T	Likely Benign		Conflicting		2	6-33443790-G-A	17	1.06e-5	-3.928	Likely Benign	0.133	Likely Benign	Likely Benign	0.144	Likely Benign										-0.19	Neutral	0.253	Benign	0.042	Benign	4.10	Benign	0.60	Tolerated	3.77	5	1	0	-2.5	30.03
c.3238G>T	A1080S	Likely Benign		Uncertain		1	6-33443790-G-T	1	6.26e-7	-3.277	Likely Benign	0.108	Likely Benign	Likely Benign	0.103	Likely Benign										0.01	Neutral	0.702	Possibly Damaging	0.346	Benign	4.16	Benign	0.08	Tolerated	3.77	5	1	1	-2.6	16.00
c.3254G>A	R1085Q	Likely Benign		Uncertain		1	6-33443806-G-A	5	3.16e-6	-3.843	Likely Benign	0.589	Likely Pathogenic	Likely Benign	0.224	Likely Benign										-1.43	Neutral	0.998	Probably Damaging	0.988	Probably Damaging	2.73	Benign	0.02	Affected	3.77	5	1	1	1.0	-28.06
c.3293G>A	S1098N	Likely Benign		Conflicting		2	6-33443845-G-A	6	3.89e-6	-5.120	Likely Benign	0.156	Likely Benign	Likely Benign	0.063	Likely Benign										-0.58	Neutral	0.369	Benign	0.120	Benign	2.76	Benign	0.36	Tolerated	3.77	5	1	1	-2.7	27.03
c.3304G>A	A1102T	Likely Benign		Uncertain		1	6-33443856-G-A	11	7.17e-6	-3.540	Likely Benign	0.070	Likely Benign	Likely Benign	0.044	Likely Benign										-0.30	Neutral	0.001	Benign	0.001	Benign	2.32	Pathogenic	0.95	Tolerated	3.77	5	1	0	-2.5	30.03
c.3314G>A	R1105Q	Likely Benign		Uncertain		2	6-33443866-G-A	3	1.96e-6	-3.666	Likely Benign	0.216	Likely Benign	Likely Benign	0.104	Likely Benign										-1.21	Neutral	0.958	Probably Damaging	0.194	Benign	2.50	Benign	0.16	Tolerated	3.77	5	1	1	1.0	-28.06
c.3328A>G	S1110G	Likely Benign		Likely Benign		1				-4.674	Likely Benign	0.079	Likely Benign	Likely Benign	0.035	Likely Benign										-2.26	Neutral	0.036	Benign	0.026	Benign	2.19	Pathogenic	0.08	Tolerated	4.32	2	1	0	0.4	-30.03
c.335G>C	G112A	Likely Benign		Uncertain		1	6-33432200-G-C	15	9.30e-6	-2.456	Likely Benign	0.119	Likely Benign	Likely Benign	0.114	Likely Benign										-2.34	Neutral	0.231	Benign	0.054	Benign	4.07	Benign	0.00	Affected	3.61	5	1	0	2.2	14.03
c.3364G>A	G1122S	Likely Benign		Benign/Likely benign		2	6-33443916-G-A	27	1.79e-5	-4.880	Likely Benign	0.072	Likely Benign	Likely Benign	0.189	Likely Benign										-0.08	Neutral	0.022	Benign	0.006	Benign	4.89	Benign	0.92	Tolerated	3.77	5	1	0	-0.4	30.03
c.3368G>A	G1123D			Uncertain		1	6-33443920-G-A	2	1.33e-6	-10.321	Likely Pathogenic	0.405	Ambiguous	Likely Benign	0.360	Likely Benign										-0.78	Neutral	0.500	Possibly Damaging	0.157	Benign	4.34	Benign	0.19	Tolerated	3.77	5	1	-1	-3.1	58.04
c.3374G>C	G1125A	Likely Benign		Uncertain		1	6-33443926-G-C	1	6.68e-7	-6.569	Likely Benign	0.083	Likely Benign	Likely Benign	0.232	Likely Benign										-0.60	Neutral	0.999	Probably Damaging	0.995	Probably Damaging	4.60	Benign	0.11	Tolerated	3.77	5	1	0	2.2	14.03
c.3377G>A	G1126D			Uncertain		1				-8.888	Likely Pathogenic	0.432	Ambiguous	Likely Benign	0.376	Likely Benign										-0.65	Neutral	0.906	Possibly Damaging	0.473	Possibly Damaging	4.82	Benign	0.02	Affected	3.77	5	1	-1	-3.1	58.04
c.3380G>C	G1127A	Likely Benign		Conflicting		4	6-33443932-G-C	4	2.68e-6	-5.949	Likely Benign	0.080	Likely Benign	Likely Benign	0.164	Likely Benign										-0.43	Neutral	0.001	Benign	0.002	Benign	4.83	Benign	1.00	Tolerated	4.32	4	1	0	2.2	14.03
c.3394T>C	S1132P	Likely Benign		Conflicting		3	6-33443946-T-C	1	6.74e-7	-1.423	Likely Benign	0.144	Likely Benign	Likely Benign	0.301	Likely Benign										0.38	Neutral	0.003	Benign	0.006	Benign	5.40	Benign	0.28	Tolerated	4.32	4	1	-1	-0.8	10.04
c.3405G>C	K1135N	Likely Benign		Uncertain		1				-5.715	Likely Benign	0.960	Likely Pathogenic	Likely Pathogenic	0.166	Likely Benign										-0.97	Neutral	0.411	Benign	0.321	Benign	5.43	Benign	0.07	Tolerated	4.32	2	1	0	0.4	-14.07
c.3434A>G	N1145S	Likely Benign		Uncertain		1	6-33444469-A-G	2	1.24e-6	-0.989	Likely Benign	0.126	Likely Benign	Likely Benign	0.308	Likely Benign										-1.15	Neutral	0.997	Probably Damaging	0.989	Probably Damaging	5.55	Benign	0.89	Tolerated	4.32	4	1	1	2.7	-27.03
c.3484C>T	P1162S	Likely Benign		Uncertain		1				-2.118	Likely Benign	0.913	Likely Pathogenic	Ambiguous	0.215	Likely Benign										-1.93	Neutral	1.000	Probably Damaging	0.999	Probably Damaging	2.73	Benign	0.55	Tolerated	3.88	3	1	-1	0.8	-10.04
c.3487C>G	H1163D			Uncertain		1				-2.107	Likely Benign	0.949	Likely Pathogenic	Ambiguous	0.476	Likely Benign										-2.60	Deleterious	0.991	Probably Damaging	0.991	Probably Damaging	5.44	Benign	0.31	Tolerated	3.88	3	1	-1	-0.3	-22.05
c.3508A>G	S1170G	Likely Benign	Coiled-coil	Uncertain		1				-4.288	Likely Benign	0.221	Likely Benign	Likely Benign	0.349	Likely Benign										-0.81	Neutral	0.241	Benign	0.229	Benign	5.31	Benign	0.54	Tolerated	4.32	4	1	0	0.4	-30.03
c.3511G>A	A1171T	Likely Benign	Coiled-coil	Uncertain		1				-3.658	Likely Benign	0.149	Likely Benign	Likely Benign	0.201	Likely Benign										-0.48	Neutral	0.245	Benign	0.138	Benign	5.45	Benign	0.07	Tolerated	4.32	4	1	0	-2.5	30.03
c.3572G>A	R1191Q	Likely Benign	Coiled-coil	Uncertain		2	6-33444607-G-A	9	5.58e-6	-1.069	Likely Benign	0.943	Likely Pathogenic	Ambiguous	0.343	Likely Benign										-1.41	Neutral	0.998	Probably Damaging	0.992	Probably Damaging	2.68	Benign	0.08	Tolerated	3.82	4	1	1	1.0	-28.06
c.3607C>G	H1203D	Likely Benign	Coiled-coil	Uncertain		1				-6.729	Likely Benign	0.525	Ambiguous	Likely Benign	0.403	Likely Benign										-1.89	Neutral	0.473	Possibly Damaging	0.265	Benign	5.51	Benign	0.24	Tolerated	3.77	5	1	-1	-0.3	-22.05
c.3631A>G	M1211V	Likely Benign	Coiled-coil	Benign		1	6-33446623-A-G	3	1.86e-6	-2.101	Likely Benign	0.258	Likely Benign	Likely Benign	0.412	Likely Benign										-0.29	Neutral	0.932	Possibly Damaging	0.949	Probably Damaging	5.43	Benign	0.72	Tolerated	3.77	5	1	2	2.3	-32.06
c.3633G>A	M1211I	Likely Benign	Coiled-coil	Uncertain		1	6-33446625-G-A	3	1.86e-6	-1.537	Likely Benign	0.764	Likely Pathogenic	Likely Benign	0.298	Likely Benign										-0.42	Neutral	0.969	Probably Damaging	0.968	Probably Damaging	5.40	Benign	1.00	Tolerated	3.77	5	1	2	2.6	-18.03
c.3638A>G	N1213S	Likely Benign	Coiled-coil	Benign		1	6-33446630-A-G	13	8.05e-6	-4.086	Likely Benign	0.081	Likely Benign	Likely Benign	0.094	Likely Benign										-0.56	Neutral	0.906	Possibly Damaging	0.551	Possibly Damaging	2.82	Benign	0.68	Tolerated	3.77	5	1	1	2.7	-27.03																10.1016/j.ajhg.2020.11.011
c.3662G>A	R1221Q	Likely Benign	Coiled-coil	Conflicting		2	6-33446654-G-A	4	2.48e-6	-5.491	Likely Benign	0.115	Likely Benign	Likely Benign	0.078	Likely Benign										-1.46	Neutral	0.836	Possibly Damaging	0.153	Benign	2.56	Benign	0.12	Tolerated	3.77	5	1	1	1.0	-28.06
c.3705G>A	M1235I	Likely Benign	Coiled-coil	Uncertain		1				-4.312	Likely Benign	0.310	Likely Benign	Likely Benign	0.027	Likely Benign										-1.44	Neutral	0.139	Benign	0.056	Benign	2.69	Benign	0.04	Affected	3.77	5	1	2	2.6	-18.03
c.3731G>A	S1244N	Likely Pathogenic	Coiled-coil	Uncertain		1				-9.008	Likely Pathogenic	0.751	Likely Pathogenic	Likely Benign	0.154	Likely Benign										-1.87	Neutral	0.997	Probably Damaging	0.992	Probably Damaging	2.10	Pathogenic	0.15	Tolerated	3.77	5	1	1	-2.7	27.03
c.373C>T	P125S	Likely Benign		Uncertain		1				-3.769	Likely Benign	0.238	Likely Benign	Likely Benign	0.121	Likely Benign										-3.57	Deleterious	0.580	Possibly Damaging	0.140	Benign	2.86	Benign	0.02	Affected	3.61	5	1	-1	0.8	-10.04
c.3773A>G	Q1258R	Likely Pathogenic	Coiled-coil	Uncertain		1				-10.971	Likely Pathogenic	0.931	Likely Pathogenic	Ambiguous	0.316	Likely Benign										-3.19	Deleterious	0.994	Probably Damaging	0.988	Probably Damaging	2.00	Pathogenic	0.00	Affected			1	1	-1.0	28.06
c.380G>A	R127Q	Likely Benign		Uncertain		1	6-33432245-G-A	6	3.72e-6	-1.711	Likely Benign	0.320	Likely Benign	Likely Benign	0.037	Likely Benign										-1.04	Neutral	0.006	Benign	0.001	Benign	4.04	Benign	0.02	Affected	3.74	4	1	1	1.0	-28.06
c.3824G>A	R1275Q	Likely Benign		Uncertain		1	6-33447872-G-A	2	1.29e-6	-4.928	Likely Benign	0.121	Likely Benign	Likely Benign	0.103	Likely Benign										-1.72	Neutral	0.898	Possibly Damaging	0.147	Benign	2.59	Benign	0.03	Affected	3.77	5	1	1	1.0	-28.06
c.3835G>A	A1279T	Likely Benign		Uncertain		2	6-33447883-G-A	2	1.29e-6	-4.871	Likely Benign	0.071	Likely Benign	Likely Benign	0.178	Likely Benign										-0.30	Neutral	0.001	Benign	0.000	Benign	2.71	Benign	0.09	Tolerated	3.77	5	1	0	-2.5	30.03
c.3862A>G	K1288E			Uncertain		1	6-33447910-A-G	5	3.22e-6	-2.751	Likely Benign	0.407	Ambiguous	Likely Benign	0.185	Likely Benign										-3.27	Deleterious	0.979	Probably Damaging	0.973	Probably Damaging	2.13	Pathogenic	0.00	Affected	3.77	5	1	0	0.4	0.94
c.3907G>A	G1303S	Likely Benign		Uncertain		1				-2.271	Likely Benign	0.125	Likely Benign	Likely Benign	0.155	Likely Benign										-0.19	Neutral	0.649	Possibly Damaging	0.433	Benign	2.84	Benign	0.18	Tolerated			1	0	-0.4	30.03
c.3913A>G	T1305A	Likely Benign		Conflicting		4	6-33451787-A-G	30	1.86e-5	-2.692	Likely Benign	0.055	Likely Benign	Likely Benign	0.069	Likely Benign										1.74	Neutral	0.000	Benign	0.001	Benign	3.24	Benign	1.00	Tolerated	3.77	5	1	0	2.5	-30.03
c.3949G>A	G1317S	Likely Benign		Conflicting		3	6-33451823-G-A	1	6.26e-7	-3.522	Likely Benign	0.145	Likely Benign	Likely Benign	0.092	Likely Benign										-2.45	Neutral	0.127	Benign	0.045	Benign	4.08	Benign	0.00	Affected	3.77	5	1	0	-0.4	30.03
c.3956C>G	A1319G	Likely Benign		Uncertain		2	6-33451830-C-G			-3.927	Likely Benign	0.084	Likely Benign	Likely Benign	0.128	Likely Benign										-0.74	Neutral	0.819	Possibly Damaging	0.581	Possibly Damaging	4.07	Benign	0.06	Tolerated	3.77	5	1	0	-2.2	-14.03
c.3958C>T	P1320S	Likely Benign		Uncertain		1	6-33451832-C-T	2	1.28e-6	-4.928	Likely Benign	0.073	Likely Benign	Likely Benign	0.097	Likely Benign										-0.69	Neutral	0.980	Probably Damaging	0.968	Probably Damaging	4.25	Benign	0.00	Affected	3.77	5	1	-1	0.8	-10.04
c.3961C>T	P1321S	Likely Benign		Uncertain		2	6-33451835-C-T	10	6.46e-6	-4.897	Likely Benign	0.077	Likely Benign	Likely Benign	0.049	Likely Benign										0.68	Neutral	0.028	Benign	0.004	Benign	4.27	Benign	0.71	Tolerated	3.77	5	1	-1	0.8	-10.04																10.1016/j.ajhg.2020.11.011
c.3964G>C	A1322P	Likely Benign		Benign		1	6-33451838-G-C			-1.153	Likely Benign	0.063	Likely Benign	Likely Benign	0.090	Likely Benign										0.03	Neutral	0.000	Benign	0.000	Benign	4.15	Benign	0.23	Tolerated	3.77	5	1	-1	-3.4	26.04
c.3970C>T	P1324S	Likely Benign		Likely Benign		1	6-33451844-C-T	5	3.26e-6	-5.451	Likely Benign	0.068	Likely Benign	Likely Benign	0.049	Likely Benign										0.35	Neutral	0.225	Benign	0.092	Benign	4.33	Benign	0.00	Affected	4.32	1	1	-1	0.8	-10.04
c.3979C>T	P1327S	Likely Benign		Uncertain		1	6-33451853-C-T			-4.744	Likely Benign	0.131	Likely Benign	Likely Benign	0.092	Likely Benign										0.28	Neutral	0.980	Probably Damaging	0.857	Possibly Damaging	4.25	Benign	0.71	Tolerated	3.77	5	1	-1	0.8	-10.04
c.3983G>A	R1328Q	Likely Benign		Uncertain		3	6-33451857-G-A	35	1.49e-4	-2.921	Likely Benign	0.273	Likely Benign	Likely Benign	0.043	Likely Benign										-1.02	Neutral	0.799	Possibly Damaging	0.098	Benign	4.12	Benign	0.03	Affected	3.77	5	1	1	1.0	-28.06
c.4000A>G	N1334D			Uncertain		1	6-33451874-A-G			-4.584	Likely Benign	0.674	Likely Pathogenic	Likely Benign	0.126	Likely Benign										-3.06	Deleterious	0.886	Possibly Damaging	0.522	Possibly Damaging	3.55	Benign	0.00	Affected	3.77	5	1	2	0.0	0.98
c.4003G>A	G1335S	Likely Pathogenic		Conflicting		2	6-33451877-G-A	3	2.37e-6	-4.495	Likely Benign	0.986	Likely Pathogenic	Likely Pathogenic	0.362	Likely Benign										-3.79	Deleterious	1.000	Probably Damaging	0.997	Probably Damaging	2.04	Pathogenic	0.00	Affected	3.77	5	1	0	-0.4	30.03
c.4013G>A	R1338Q	Likely Benign		Conflicting		3	6-33451887-G-A	12	8.40e-6	-3.494	Likely Benign	0.317	Likely Benign	Likely Benign	0.076	Likely Benign										-1.87	Neutral	0.896	Possibly Damaging	0.194	Benign	3.81	Benign	0.02	Affected	3.77	5	1	1	1.0	-28.06
c.401G>A	S134N	Likely Benign		Uncertain		1				-5.534	Likely Benign	0.813	Likely Pathogenic	Ambiguous	0.075	Likely Benign										-1.62	Neutral	0.001	Benign	0.002	Benign	3.90	Benign	0.00	Affected	3.61	5	1	1	-2.7	27.03
c.4021G>A	A1341T	Likely Benign		Conflicting		3	6-33451895-G-A	45	3.44e-5	-3.224	Likely Benign	0.081	Likely Benign	Likely Benign	0.099	Likely Benign										-0.58	Neutral	0.000	Benign	0.000	Benign	4.09	Benign	0.03	Affected	3.77	5	1	0	-2.5	30.03
c.4021G>T	A1341S	Likely Benign		Uncertain		1	6-33451895-G-T			-2.867	Likely Benign	0.078	Likely Benign	Likely Benign	0.099	Likely Benign										0.80	Neutral	0.000	Benign	0.001	Benign	4.40	Benign	1.00	Tolerated	3.77	5	1	1	-2.6	16.00
c.404G>A	R135Q			Uncertain		1	6-33432701-G-A	5	3.84e-6	-8.011	Likely Pathogenic	0.853	Likely Pathogenic	Ambiguous	0.087	Likely Benign										-1.94	Neutral	0.327	Benign	0.100	Benign	3.76	Benign	0.02	Affected	3.61	5	1	1	1.0	-28.06
c.407G>A	R136Q			Benign		1	6-33432704-G-A	13	9.17e-6	-11.146	Likely Pathogenic	0.950	Likely Pathogenic	Ambiguous	0.190	Likely Benign										-2.26	Neutral	0.957	Probably Damaging	0.342	Benign	3.52	Benign	0.01	Affected	3.61	5	1	1	1.0	-28.06
c.416G>A	S139N	Likely Benign		Uncertain		1	6-33432713-G-A	3	2.22e-6	-4.584	Likely Benign	0.688	Likely Pathogenic	Likely Benign	0.109	Likely Benign										-0.75	Neutral	0.149	Benign	0.047	Benign	4.14	Benign	0.24	Tolerated	3.61	5	1	1	-2.7	27.03
c.43G>A	A15T	Likely Benign		Uncertain		1	6-33420307-G-A	4	2.60e-6	-3.720	Likely Benign	0.125	Likely Benign	Likely Benign	0.086	Likely Benign										-0.08	Neutral	0.602	Possibly Damaging	0.017	Benign	4.16	Benign	0.00	Affected	4.32	1	1	0	-2.5	30.03
c.43G>C	A15P	Likely Benign		Uncertain		1				-3.436	Likely Benign	0.097	Likely Benign	Likely Benign	0.146	Likely Benign										-0.23	Neutral	0.880	Possibly Damaging	0.123	Benign	4.09	Benign	0.00	Affected			1	-1	-3.4	26.04
c.455G>A	R152Q			Uncertain		1	6-33432752-G-A	5	3.14e-6	-10.336	Likely Pathogenic	0.989	Likely Pathogenic	Likely Pathogenic	0.181	Likely Benign										-2.34	Neutral	0.997	Probably Damaging	0.968	Probably Damaging	3.89	Benign	0.00	Affected	3.61	5	1	1	1.0	-28.06
c.491G>A	R164Q			Uncertain		1	6-33432788-G-A	2	1.24e-6	-11.208	Likely Pathogenic	0.600	Likely Pathogenic	Likely Benign	0.184	Likely Benign										-1.86	Neutral	0.957	Probably Damaging	0.342	Benign	3.82	Benign	0.00	Affected	3.74	4	1	1	1.0	-28.06
c.509G>A	R170Q			Pathogenic/Likely path.		6				-9.021	Likely Pathogenic	0.798	Likely Pathogenic	Ambiguous	0.221	Likely Benign										-2.31	Neutral	0.947	Possibly Damaging	0.342	Benign	3.91	Benign	0.00	Affected	3.74	4	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.515G>A	R172Q			Uncertain		1	6-33435157-G-A	3	1.86e-6	-7.245	In-Between	0.465	Ambiguous	Likely Benign	0.135	Likely Benign										-1.72	Neutral	0.804	Possibly Damaging	0.091	Benign	4.04	Benign	0.04	Affected	3.61	5	1	1	1.0	-28.06
c.583G>C	A195P	Likely Pathogenic		Likely Pathogenic		1				-9.715	Likely Pathogenic	0.978	Likely Pathogenic	Likely Pathogenic	0.152	Likely Benign										-3.03	Deleterious	0.997	Probably Damaging	0.916	Probably Damaging	4.00	Benign	0.04	Affected	3.54	6	1	-1	-3.4	26.04
c.597C>A	N199K (3D Viewer)		PH	Uncertain		1				-8.198	Likely Pathogenic	0.686	Likely Pathogenic	Likely Benign	0.024	Likely Benign	-0.19	Likely Benign	0.1	0.03	Likely Benign	-0.08	Likely Benign	0.33	Likely Benign	-1.48	Neutral	0.276	Benign	0.083	Benign	4.27	Benign	0.13	Tolerated	3.47	9	1	0	-0.4	14.07	207.8	21.5	-0.1	1.5	0.1	0.0						X		Uncertain	Asn199, located in the N-terminal loop before the first anti-parallel β sheet strand (res. Ile205-Pro208), is replaced by a positively charged lysine. On the protein surface, both the carboxamide group of Asn199 and the amino group of Lys199 side chains can form hydrogen bonds with the backbone carbonyl groups of residues (e.g., Ala249) at the end of an α helix (res. Ala236-Lys251). However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.5G>A	S2N	Likely Benign		Uncertain		2	6-33420269-G-A	3	1.96e-6	-4.104	Likely Benign	0.207	Likely Benign	Likely Benign	0.092	Likely Benign										-0.36	Neutral	0.000	Benign	0.000	Benign	4.06	Benign	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.68A>G	D23G	Likely Benign		Uncertain		1				-2.622	Likely Benign	0.684	Likely Pathogenic	Likely Benign	0.100	Likely Benign										-2.45	Neutral	0.805	Possibly Damaging	0.539	Possibly Damaging	3.50	Benign	0.00	Affected			1	-1	3.1	-58.04
c.719A>G	D240G	Likely Pathogenic	PH	Uncertain		1				-12.825	Likely Pathogenic	0.951	Likely Pathogenic	Ambiguous	0.912	Likely Pathogenic	1.85	Ambiguous	0.1	2.72	Destabilizing	2.29	Destabilizing	0.24	Likely Benign	-6.19	Deleterious	0.993	Probably Damaging	0.984	Probably Damaging	5.79	Benign	0.01	Affected			1	-1	3.1	-58.04
c.74G>A	R25Q	Likely Benign		Uncertain		1	6-33423483-G-A	15	9.29e-6	-4.126	Likely Benign	0.212	Likely Benign	Likely Benign	0.038	Likely Benign										-0.70	Neutral	0.829	Possibly Damaging	0.614	Possibly Damaging	4.01	Benign	0.00	Affected	4.32	1	1	1	1.0	-28.06
c.667A>G	T223A (3D Viewer)		PH	Uncertain		1	6-33435518-A-G	3	1.86e-6	-7.076	In-Between	0.316	Likely Benign	Likely Benign	0.574	Likely Pathogenic	0.30	Likely Benign	0.1	0.77	Ambiguous	0.54	Ambiguous	0.74	Ambiguous	-3.36	Deleterious	0.231	Benign	0.058	Benign	5.74	Benign	0.09	Tolerated	3.41	13	1	0	2.5	-30.03	186.4	44.0	0.0	0.0	0.0	0.0	X	X						Uncertain	The introduced residue Ala223 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Unlike the hydroxyl group of the Thr223 side chain in the WT protein, the methyl side chain of Ala223 cannot form hydrogen bonds with nearby residues Thr228 and Lys207. Without these hydrogen-bonding interactions at the β sheet surface, the secondary structure element becomes unstable and partially unfolds in the variant simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.667A>T	T223S (3D Viewer)		PH	Conflicting		2	6-33435518-A-T	3	1.86e-6	-7.714	In-Between	0.410	Ambiguous	Likely Benign	0.535	Likely Pathogenic	0.26	Likely Benign	0.1	0.50	Ambiguous	0.38	Likely Benign	0.62	Ambiguous	-2.86	Deleterious	0.421	Benign	0.058	Benign	5.80	Benign	0.02	Affected	3.41	13	1	1	-0.1	-14.03	200.7	17.3	-0.2	0.2	0.0	0.0						X		Uncertain	The introduced residue Ser223 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Its hydroxyl group forms hydrogen bonds with nearby residues Thr228 and Lys207 in the variant simulations, similar to the hydroxyl group of Thr223 in the WT simulations. These hydrogen-bonding interactions at the β sheet surface contribute to the stability of the secondary structure element and may prevent it from unfolding. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.670A>G	T224A (3D Viewer)		PH	Uncertain		3	6-33435521-A-G	2	1.24e-6	-7.379	In-Between	0.651	Likely Pathogenic	Likely Benign	0.464	Likely Benign	0.33	Likely Benign	0.1	1.05	Ambiguous	0.69	Ambiguous	0.91	Ambiguous	-2.96	Deleterious	0.243	Benign	0.079	Benign	5.57	Benign	0.57	Tolerated	3.41	13	1	0	2.5	-30.03	169.0	41.4	-0.5	1.1	-0.4	0.0	X	X						Uncertain	The introduced residue Ala224 is located on the outer surface of an anti-parallel β sheet strand (res. Cys219-Thr224). Unlike the hydroxyl group of the Thr224 side chain in the WT model, the methyl side chain of Ala224 cannot form hydrogen bonds with nearby residues Ser204, Ser226, and Gly227. Without these hydrogen-bonding interactions at the β sheet surface, the secondary structure element becomes unstable and unfolds during the variant simulations. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.767A>G	N256S (3D Viewer)	Likely Pathogenic	C2	Likely Pathogenic		1				-10.640	Likely Pathogenic	0.950	Likely Pathogenic	Ambiguous	0.707	Likely Pathogenic	0.31	Likely Benign	0.2	0.36	Likely Benign	0.34	Likely Benign	0.48	Likely Benign	-4.33	Deleterious	0.997	Probably Damaging	0.970	Probably Damaging	5.87	Benign	0.02	Affected	3.39	15	1	1	2.7	-27.03
c.82T>C	S28P	Likely Benign		Uncertain		1				-3.309	Likely Benign	0.051	Likely Benign	Likely Benign	0.047	Likely Benign										1.37	Neutral	0.000	Benign	0.000	Benign	4.53	Benign	0.00	Affected	4.32	1	1	-1	-0.8	10.04
c.694G>A	A232T (3D Viewer)		PH	Benign		1	6-33435545-G-A	1	6.20e-7	-7.655	In-Between	0.874	Likely Pathogenic	Ambiguous	0.469	Likely Benign	0.47	Likely Benign	0.1	-0.04	Likely Benign	0.22	Likely Benign	0.61	Ambiguous	-1.42	Neutral	0.608	Possibly Damaging	0.240	Benign	5.80	Benign	0.09	Tolerated	3.40	14	1	0	-2.5	30.03	210.8	-42.0	0.5	0.1	0.4	0.5						X		Uncertain	The hydroxyl group of Thr232, located at the end of an anti-parallel β sheet strand (res. Thr228-Ala232), forms hydrogen bonds with nearby residues Glu217, Cys233, and Cys219 in the variant simulations. These hydrogen-bonding interactions at the β sheet surface contribute to the stability of the secondary structure element and prevent it from unfolding. The new hydrogen bond interactions may be more favorable for structural stability than the steric interactions of the methyl side chain of Ala with the side chains of Gln216 and Cys219 in the WT. However, since the model ends abruptly at the N-terminus, no definite conclusions can be drawn from the simulations.
c.703T>C	S235P (3D Viewer)	Likely Pathogenic	PH	Likely Pathogenic		1				-14.857	Likely Pathogenic	0.998	Likely Pathogenic	Likely Pathogenic	0.870	Likely Pathogenic	4.02	Destabilizing	0.1	6.91	Destabilizing	5.47	Destabilizing	1.23	Destabilizing	-4.24	Deleterious	0.917	Possibly Damaging	0.446	Benign	5.47	Benign	0.01	Affected	3.40	14	1	-1	-0.8	10.04	201.5	17.0	0.1	0.0	-0.6	0.0		X						Potentially Pathogenic	In the WT, the hydroxyl group of Ser235, located in a β-α loop between an anti-parallel β sheet strand (res. Gly227-Phe231) and an α helix (residues Ala236-Val250), forms hydrogen bonds with the GAP domain loop residue Glu680 and with the backbone amide groups of Ala237 and Glu238 from the α helix. In the variant simulations, the pyrrolidine ring of Pro235 cannot stabilize the α helix end or maintain tertiary bonding interactions between the PH and GAP domains via hydrogen bonding as effectively as serine.
c.862G>A	D288N (3D Viewer)	Likely Pathogenic	C2	Uncertain		1	6-33437767-G-A	2	1.24e-6	-10.535	Likely Pathogenic	0.521	Ambiguous	Likely Benign	0.321	Likely Benign	-0.39	Likely Benign	0.1	0.01	Likely Benign	-0.19	Likely Benign	-0.03	Likely Benign	-3.73	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.78	Pathogenic	0.05	Affected	3.38	23	1	2	0.0	-0.98
c.910G>A	D304N (3D Viewer)		C2	Uncertain		1				-6.194	Likely Benign	0.391	Ambiguous	Likely Benign	0.345	Likely Benign	0.30	Likely Benign	0.1	-0.08	Likely Benign	0.11	Likely Benign	0.21	Likely Benign	-4.18	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	1.81	Pathogenic	0.03	Affected	3.38	23	1	2	0.0	-0.98
c.745G>A	A249T (3D Viewer)	Likely Benign	PH	Uncertain		1				-3.564	Likely Benign	0.805	Likely Pathogenic	Ambiguous	0.487	Likely Benign	1.50	Ambiguous	0.6	1.39	Ambiguous	1.45	Ambiguous	0.30	Likely Benign	-0.96	Neutral	0.990	Probably Damaging	0.815	Possibly Damaging	5.65	Benign	0.40	Tolerated	3.39	15	1	0	-2.5	30.03	214.5	-43.3	0.0	0.0	0.5	0.2						X		Potentially Benign	The methyl group of Ala249, located on the surface of an α helix (res. Ala236-Val250) facing an anti-parallel β sheet strand (res. Ile205-Val209), packs against nearby hydrophobic residues such as Leu200, Leu246, and Val250. In the variant simulations, the hydroxyl group of Thr249, which is not suitable for hydrophobic packing, forms a stable hydrogen bond with the backbone carbonyl of Asn245 in the same helix. Although this interaction could theoretically weaken the structural integrity of the α helix, this destabilizing effect is not observed in the variant simulations.
c.762G>C	K254N (3D Viewer)	Likely Pathogenic	PH	Uncertain		1				-13.306	Likely Pathogenic	0.999	Likely Pathogenic	Likely Pathogenic	0.757	Likely Pathogenic	0.73	Ambiguous	0.2	1.87	Ambiguous	1.30	Ambiguous	1.19	Destabilizing	-4.23	Deleterious	0.384	Benign	0.070	Benign	5.93	Benign	0.01	Affected	3.39	15	1	0	0.4	-14.07	215.3	-21.0	-1.0	1.7	0.2	0.3		X						Potentially Pathogenic	The amino group of Lys254, located in an α-β loop connecting the PH and C2 domains (res. Lys251-Arg258), forms salt bridges with the carboxylate groups of Glu244 and Asp684. Since the neutral carboxamide group of the Asn254 side chain cannot form salt bridges with acidic residues, the residue swap potentially weakens the tertiary structure assembly and/or influences the loop positioning. Regardless, in both the variant and WT simulations, all hydrogen bonds formed by the residue’s side chain were broken, and the residue rotated outwards. The partially α helical conformation of the loop, which extends to a nearby α helix (res. Met414-Asn426), is dynamic, making it unclear if the mutation affects it.