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.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.1723C>T	R575C (3D Viewer)	Likely Pathogenic	GAP	Conflicting		3	6-33440775-C-T	23	1.43e-5	-11.179	Likely Pathogenic	0.630	Likely Pathogenic	Likely Benign	0.715	Likely Pathogenic	1.39	Ambiguous	0.2	0.50	Ambiguous	0.95	Ambiguous	0.73	Ambiguous	-5.43	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	-1.30	Pathogenic	0.02	Affected	3.37	35	-4	-3	7.0	-53.05	227.7	99.2	0.0	0.0	0.0	0.1		X						Potentially Pathogenic	The guanidinium group of Arg575, located in an α-helix (res. Arg563-Glu578), forms salt bridges with the carboxylate groups of Asp463 and Asp467, and it also hydrogen bonds with the hydroxyl group of Ser466 on an opposing α-helix (res. Ala461-Phe476) in the WT simulations. In the variant simulations, the thiol group of the Cys575 side chain, which is neither positively charged nor particularly hydrophilic, packs against the hydrophobic Met470 on an opposing α-helix (res. Ala461-Arg475). Additionally, although the thiol group is not an effective hydrogen bonder, the Cys575 side chain rotates to hydrogen bond with the backbone carbonyl group of Ser571 in the same α-helix, which could theoretically lower the helix integrity. Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.
c.1724G>A	R575H (3D Viewer)		GAP	Conflicting		4	6-33440776-G-A	204	1.27e-4	-11.142	Likely Pathogenic	0.496	Ambiguous	Likely Benign	0.707	Likely Pathogenic	0.81	Ambiguous	0.2	-0.22	Likely Benign	0.30	Likely Benign	1.31	Destabilizing	-2.34	Neutral	1.000	Probably Damaging	0.998	Probably Damaging	-1.33	Pathogenic	0.05	Affected	3.37	35	2	0	1.3	-19.05	244.7	80.6	0.0	0.0	0.3	0.0		X						Potentially Pathogenic	The guanidinium group of Arg575, located in an α-helix (res. Arg563-Glu578), forms salt bridges with the carboxylate groups of Asp463 and Asp467, and it also hydrogen bonds with the hydroxyl group of Ser466 on an opposing α-helix (res. Ala461-Phe476) in the WT simulations. In the variant simulations, the imidazole ring of His575 (in its neutral epsilon protonated form) cannot form the same salt bridges as the guanidinium group of the non-mutated Arg575. Instead, His575 only forms weak hydrogen bonds with the hydroxyl groups of Ser466 and Ser571. Overall, the residue swap has the potential to substantially affect the tertiary structure assembly during the protein folding process.
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.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.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.2224C>T	R742W	Likely Benign		Uncertain		1	6-33441689-C-T	6	3.72e-6	-7.725	In-Between	0.133	Likely Benign	Likely Benign	0.079	Likely Benign										-1.71	Neutral	0.992	Probably Damaging	0.684	Possibly Damaging	2.66	Benign	0.01	Affected	4.32	2	-3	2	3.6	30.03
c.2239G>C	V747L	Likely Benign		Uncertain		1	6-33441704-G-C	2	1.24e-6	-2.790	Likely Benign	0.096	Likely Benign	Likely Benign	0.047	Likely Benign										-0.52	Neutral	0.065	Benign	0.033	Benign	2.67	Benign	0.00	Affected	4.32	2	2	1	-0.4	14.03
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.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.2243T>G	L748R	Likely Benign		Conflicting		2	6-33441708-T-G	3	1.86e-6	-3.331	Likely Benign	0.245	Likely Benign	Likely Benign	0.055	Likely Benign										-0.67	Neutral	0.912	Possibly Damaging	0.448	Possibly Damaging	2.73	Benign	0.02	Affected	4.32	2	-3	-2	-8.3	43.03
c.2245C>T	R749W			Likely Benign		1	6-33441710-C-T	3	1.86e-6	-7.647	In-Between	0.338	Likely Benign	Likely Benign	0.173	Likely Benign										-2.62	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.59	Benign	0.00	Affected	4.32	2	2	-3	3.6	30.03
c.2246G>A	R749Q	Likely Benign		Likely Benign		1	6-33441711-G-A	4	2.48e-6	-3.069	Likely Benign	0.212	Likely Benign	Likely Benign	0.152	Likely Benign										-1.00	Neutral	0.999	Probably Damaging	0.994	Probably Damaging	2.64	Benign	0.03	Affected	4.32	2	1	1	1.0	-28.06
c.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.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.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.1786C>T	R596C (3D Viewer)	Likely Pathogenic	GAP	Conflicting		2	6-33440838-C-T	6	3.72e-6	-10.805	Likely Pathogenic	0.972	Likely Pathogenic	Likely Pathogenic	0.633	Likely Pathogenic	2.94	Destabilizing	0.0	1.49	Ambiguous	2.22	Destabilizing	-0.03	Likely Benign	-7.96	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.41	Pathogenic	0.00	Affected	3.37	35	-4	-3	7.0	-53.05	230.7	97.9	-0.1	0.0	-0.3	0.4		X		X				Potentially Pathogenic	The guanidinium group of Arg596, located in an α helix (res. Glu582-Met603), forms a salt bridge with the carboxylate group of Glu495 from another α helix (res. Leu489-Glu519). In the WT simulations, the side chain of Arg596 hydrogen bonds with the backbone carbonyl groups of Asn487, Glu486, Arg485, and Phe484. Additionally, Arg596 can hydrogen bond with the carboxamide group of the Asn487 side chain on an opposing loop that links two α helices (res. Ala461-Arg475, res. Leu489-Glu519).In the variant simulations, the thiol group of the Cys596 side chain is unable to form salt bridges or any of the hydrogen bonds that the Arg596 side chain can. Thus, the residue swap could affect the tertiary structure assembly more profoundly than observed in the simulations. Notably, Arg596 plays a key role in positioning the aforementioned loop, which is crucial for the placement of the “arginine finger” or the Arg485 side chain during RasGTPase activation.
c.2275A>C	M759L	Likely Benign		Uncertain		1	6-33441740-A-C	2	1.24e-6	-2.431	Likely Benign	0.093	Likely Benign	Likely Benign	0.048	Likely Benign										-0.53	Neutral	0.002	Benign	0.005	Benign	2.84	Benign	1.00	Tolerated	3.99	5	4	2	1.9	-18.03
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.227C>G	S76C	Likely Benign		Uncertain		1	6-33425835-C-G	2	1.24e-6	-5.408	Likely Benign	0.100	Likely Benign	Likely Benign	0.076	Likely Benign										-1.78	Neutral	0.992	Probably Damaging	0.869	Possibly Damaging	3.71	Benign	0.00	Affected	4.32	1	0	-1	3.3	16.06
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.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>A	R596H (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33440839-G-A	15	9.29e-6	-11.128	Likely Pathogenic	0.950	Likely Pathogenic	Ambiguous	0.717	Likely Pathogenic	3.00	Destabilizing	0.9	0.43	Likely Benign	1.72	Ambiguous	1.35	Destabilizing	-4.97	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.43	Pathogenic	0.00	Affected	3.37	35	2	0	1.3	-19.05	223.5	80.5	-0.1	0.0	-0.1	0.3		X		X				Potentially Pathogenic	The guanidinium group of Arg596, located in an α helix (res. Glu582-Met603), forms a salt bridge with the carboxylate group of Glu495 from another α helix (res. Leu489-Glu519). In the WT simulations, the side chain of Arg596 hydrogen bonds with the backbone carbonyl groups of Asn487, Glu486, Arg485, and Phe484. Additionally, Arg596 can hydrogen bond with the carboxamide group of the Asn487 side chain on an opposing loop that links two α helices (res. Ala461-Arg475, res. Leu489-Glu519).In the variant simulations, the imidazole ring of His596 can form hydrogen bonds with the same residues as arginine; however, these interactions are not as coordinated or strong in comparison. Thus, the residue swap could affect the tertiary structure assembly more profoundly than observed in the simulations. Notably, Arg596 plays a key role in positioning the aforementioned loop, which is crucial for the placement of the “arginine finger” or the Arg485 side chain during RasGTPase activation.
c.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>A	D768N	Likely Benign		Uncertain		1	6-33442460-G-A	2	2.57e-6	-6.892	Likely Benign	0.453	Ambiguous	Likely Benign	0.048	Likely Benign										-0.77	Neutral	0.106	Benign	0.009	Benign	4.07	Benign	0.96	Tolerated	3.64	6	1	2	0.0	-0.98
c.2302G>T	D768Y	Likely Pathogenic		Uncertain		1	6-33442460-G-T			-9.866	Likely Pathogenic	0.824	Likely Pathogenic	Ambiguous	0.234	Likely Benign										-2.86	Deleterious	0.989	Probably Damaging	0.806	Possibly Damaging	4.01	Benign	0.07	Tolerated	3.64	6	-4	-3	2.2	48.09
c.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>A	R775Q	Likely Benign		Conflicting		3	6-33442482-G-A	11	1.41e-5	-4.476	Likely Benign	0.229	Likely Benign	Likely Benign	0.085	Likely Benign										-0.63	Neutral	0.969	Probably Damaging	0.863	Possibly Damaging	4.17	Benign	0.16	Tolerated	3.64	6	1	1	1.0	-28.06																10.1016/j.ajhg.2020.11.011
c.2324G>C	R775P	Likely Benign		Benign		1				-5.072	Likely Benign	0.452	Ambiguous	Likely Benign	0.168	Likely Benign										-0.79	Neutral	0.971	Probably Damaging	0.944	Probably Damaging	4.13	Benign	0.07	Tolerated	3.64	6	-2	0	2.9	-59.07
c.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.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.1811C>T	S604L (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1	6-33440863-C-T	6	3.72e-6	-14.683	Likely Pathogenic	0.965	Likely Pathogenic	Likely Pathogenic	0.639	Likely Pathogenic	-0.94	Ambiguous	0.1	-1.24	Ambiguous	-1.09	Ambiguous	-0.31	Likely Benign	-5.97	Deleterious	1.000	Probably Damaging	0.991	Probably Damaging	3.09	Benign	0.00	Affected	3.37	35	-3	-2	4.6	26.08	234.0	-49.6	0.0	0.1	0.3	0.5	X			X				Potentially Pathogenic	Ser604 is located in a short turn between an α helix (res. Glu582-Met603) and a short α helical section (res. Ser606-Phe608). In the WT simulations, the hydroxyl side chain of Ser604 periodically hydrogen bonds with the backbone carbonyl groups of other α helix residues (e.g., Pro600, Met603). Serine weakens the α helix secondary structure, and thus, Ser604 along with Pro605 breaks the α helix, facilitating the turn in the WT structure.In contrast, in the variant simulations, Leu604 forms a few hydrophobic interactions (e.g., Leu607, Phe608). More importantly, the helix end is more stable than with Ser604 in the WT. 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, Ser604 directly hydrogen bonds with Ras residues Ser65 and Ala66 in the WT SynGAP-Ras complex. The hydrophobic leucine cannot maintain these interactions with Ras at the GAP-Ras interface. Thus, the effect of the residue swap on the complex formation with the GTPase cannot be fully explored in the solvent-only simulations.
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.2353C>T	R785C	Likely Pathogenic	SH3-binding motif	Uncertain		1	6-33442905-C-T	29	1.80e-5	-5.887	Likely Benign	0.662	Likely Pathogenic	Likely Benign	0.126	Likely Benign										-5.06	Deleterious	0.144	Benign	0.046	Benign	2.22	Pathogenic	0.00	Affected	3.64	6	-4	-3	7.0	-53.05
c.2359C>A	P787T	Likely Pathogenic	SH3-binding motif	Likely Benign		1	6-33442911-C-A	17	1.05e-5	-4.813	Likely Benign	0.603	Likely Pathogenic	Likely Benign	0.258	Likely Benign										-4.40	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.46	Pathogenic	0.01	Affected	3.64	6	0	-1	0.9	3.99
c.2359C>T	P787S		SH3-binding motif	Uncertain		1	6-33442911-C-T	3	1.86e-6	-4.203	Likely Benign	0.564	Ambiguous	Likely Benign	0.221	Likely Benign										-3.81	Deleterious	1.000	Probably Damaging	0.999	Probably Damaging	2.48	Pathogenic	0.02	Affected	3.64	6	-1	1	0.8	-10.04
c.2369C>A	T790N		SH3-binding motif	Conflicting		3	6-33442921-C-A	69	4.28e-5	-5.243	Likely Benign	0.276	Likely Benign	Likely Benign	0.103	Likely Benign										-2.54	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.27	Pathogenic	0.02	Affected	3.64	6	0	0	-2.8	13.00
c.2369C>G	T790S	Likely Benign	SH3-binding motif	Uncertain		1				-3.914	Likely Benign	0.123	Likely Benign	Likely Benign	0.134	Likely Benign										-1.83	Neutral	0.997	Probably Damaging	0.989	Probably Damaging	2.39	Pathogenic	0.33	Tolerated	3.64	6	1	1	-0.1	-14.03
c.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.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.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.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>G	Y807C		SH3-binding motif	Uncertain		1	6-33442972-A-G	1	6.20e-7	-7.228	In-Between	0.204	Likely Benign	Likely Benign	0.243	Likely Benign										-3.89	Deleterious	0.997	Probably Damaging	0.934	Probably Damaging	2.42	Pathogenic	0.01	Affected	3.77	5	0	-2	3.8	-60.04
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.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.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.1966G>C	E656Q (3D Viewer)		GAP	Uncertain		1	6-33441225-G-C	1	6.20e-7	-9.145	Likely Pathogenic	0.766	Likely Pathogenic	Likely Benign	0.249	Likely Benign	-0.14	Likely Benign	0.0	-0.81	Ambiguous	-0.48	Likely Benign	0.25	Likely Benign	-2.29	Neutral	0.980	Probably Damaging	0.528	Possibly Damaging	3.46	Benign	0.02	Affected	3.39	24	2	2	0.0	-0.98	224.3	1.7	0.0	0.1	0.1	0.0		X						Potentially Benign	The carboxylate side chain of Glu656, located on an α helix (res. Ser641-Glu666), frequently forms a hydrogen bond with the nearby residue Ser659 on the same α helix. In the variant simulations, the carboxamide side chain of Gln656 alternatively forms a hydrogen bond with either Ser659 or Glu548 on an opposing helix (res. Ala533-Val560).Although the frequent interaction between Gln656 and Glu548 may strengthen or stabilize the tertiary structure assembly, the effect is likely to be marginal.
c.1991T>C	L664S (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441250-T-C	1	6.20e-7	-16.498	Likely Pathogenic	0.997	Likely Pathogenic	Likely Pathogenic	0.543	Likely Pathogenic	3.75	Destabilizing	0.2	3.63	Destabilizing	3.69	Destabilizing	2.77	Destabilizing	-5.99	Deleterious	1.000	Probably Damaging	0.996	Probably Damaging	2.85	Benign	0.00	Affected	3.38	28	-3	-2	-4.6	-26.08	215.5	50.1	0.0	0.0	-0.2	0.2						X		Potentially Benign	The iso-butyl side chain of L664, located on an α-helix (res. Ser641-Glu666), hydrophobically interacts with residues in the inter-helix space between three helices (res. Glu617-Asn635, res. Glu582-Met603, and res. Ser641-Glu666), such as Ile589, Phe663, and Met660. In the variant simulations, the hydroxyl group of Ser664 forms hydrogen bonds with the backbone carbonyl oxygen of another helix residue, such as Met660 or Gln661. This interaction is known to destabilize hydrogen bonding in the α-helix, but this effect was not observed in the simulations. Additionally, Ser664 occasionally forms hydrogen bonds with the carboxylate group of Asp586 on another α-helix (res. Glu582-Met603), which could minimally influence the tertiary structure assembly. Despite these interactions, no major negative effects on the protein structure were observed during the simulations.
c.1997A>G	E666G (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441256-A-G	10	6.20e-6	-12.261	Likely Pathogenic	0.911	Likely Pathogenic	Ambiguous	0.522	Likely Pathogenic	1.57	Ambiguous	0.1	1.46	Ambiguous	1.52	Ambiguous	0.93	Ambiguous	-6.25	Deleterious	1.000	Probably Damaging	0.970	Probably Damaging	3.37	Benign	0.02	Affected	3.38	28	0	-2	3.1	-72.06	173.9	98.5	0.0	0.0	-0.7	0.0		X						Potentially Pathogenic	In the WT simulations, the carboxylate group of Glu666, located on the α-helix (res. Ser641-Glu666), is involved in a highly coordinated hydrogen-bonding network between residues from two α-helices (res. Ser641-Glu666 and res. Arg563-Glu578) and from the α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), such as Lys566, Thr672, and Asn669. In the variant simulations, the carbonyl group of Gly666 occasionally forms hydrogen bonds with Lys566 and Asn669. However, Gly666 lacks a side chain and thus cannot maintain as well-coordinated a hydrogen-bond network as Glu666 in the WT, which may affect the tertiary structure assembly.
c.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.2443C>T	R815C	Likely Pathogenic	SH3-binding motif	Uncertain		1	6-33442995-C-T	5	3.10e-6	-9.373	Likely Pathogenic	0.828	Likely Pathogenic	Ambiguous	0.174	Likely Benign										-3.89	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	2.59	Benign	0.00	Affected	4.32	4	-4	-3	7.0	-53.05
c.2444G>A	R815H		SH3-binding motif	Likely Benign		2	6-33442996-G-A	24	1.49e-5	-7.474	In-Between	0.553	Ambiguous	Likely Benign	0.157	Likely Benign										-1.81	Neutral	1.000	Probably Damaging	0.998	Probably Damaging	2.61	Benign	0.02	Affected	4.32	4	2	0	1.3	-19.05																10.1016/j.ajhg.2020.11.011
c.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.2474C>T	S825L	Likely Pathogenic		Uncertain		1	6-33443026-C-T	1	6.20e-7	-4.987	Likely Benign	0.910	Likely Pathogenic	Ambiguous	0.249	Likely Benign										-4.30	Deleterious	0.999	Probably Damaging	0.994	Probably Damaging	1.94	Pathogenic	0.01	Affected	3.77	5	-2	-3	4.6	26.08
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.2493G>C	E831D	Likely Benign		Uncertain		1	6-33443045-G-C	1	6.19e-7	-3.055	Likely Benign	0.063	Likely Benign	Likely Benign	0.073	Likely Benign										1.23	Neutral	0.002	Benign	0.002	Benign	2.64	Benign	0.77	Tolerated	3.77	5	3	2	0.0	-14.03
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.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.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.2015C>T	T672M (3D Viewer)		GAP	Conflicting		2	6-33441274-C-T	19	1.18e-5	-9.472	Likely Pathogenic	0.174	Likely Benign	Likely Benign	0.127	Likely Benign	0.31	Likely Benign	0.4	1.52	Ambiguous	0.92	Ambiguous	0.41	Likely Benign	-4.34	Deleterious	0.993	Probably Damaging	0.520	Possibly Damaging	3.39	Benign	0.00	Affected	3.40	25	-1	-1	2.6	30.09	231.9	-52.9	1.1	0.1	0.5	0.0		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. Met672 can only form a hydrogen bond with the amino group of the Lys566 side chain via its backbone carbonyl group. Nevertheless, the Lys566-Glu666 salt bridge forms intermittently. This is possible because Asn669 keeps the carboxylate group of Glu666 in the vicinity through hydrogen bonding, and the hydrophobic side chain of Met stays mostly rotated away from the salt bridge. Consequently, no drastic disruption of the hydrogen-bond network that keeps the loop close to the helices occurs in the variant simulations.
c.2047A>G	I683V (3D Viewer)	Likely Benign	GAP	Uncertain		1	6-33441306-A-G	2	1.24e-6	-7.588	In-Between	0.138	Likely Benign	Likely Benign	0.112	Likely Benign	0.90	Ambiguous	0.0	0.60	Ambiguous	0.75	Ambiguous	0.76	Ambiguous	-0.78	Neutral	0.538	Possibly Damaging	0.080	Benign	3.35	Benign	0.14	Tolerated	3.42	17	4	3	-0.3	-14.03	215.6	29.1	0.0	0.0	-0.7	0.1						X		Potentially Benign	The sec-butyl side chain of Ile683, located in an entangled α-α loop connecting the two α-helices (res. Ser641-Glu666 and res. Leu685-Val699), is sterically packed against His453 and Glu688. In the variant simulations, the iso-propyl side chain of Val683 has similar size and physicochemical properties as Ile630 in the WT, and thus, it is able to maintain similar interactions in the inter-helix space. Consequently, no negative structural effects are observed during the simulations due to the residue swap.
c.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.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.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.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.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.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.2089T>C	W697R (3D Viewer)	Likely Pathogenic	GAP	Likely Benign		1	6-33441348-T-C	1	6.20e-7	-10.020	Likely Pathogenic	0.941	Likely Pathogenic	Ambiguous	0.401	Likely Benign	1.14	Ambiguous	0.1	1.18	Ambiguous	1.16	Ambiguous	1.25	Destabilizing	-9.50	Deleterious	1.000	Probably Damaging	0.994	Probably Damaging	3.45	Benign	0.02	Affected	3.46	13	2	-3	-3.6	-30.03	254.4	-41.2	0.0	0.0	-0.7	0.0						X		Potentially Benign	The indole ring of Trp697, located on the outer surface of an α-helix (res. Leu685-Val699), is not involved in any long-lasting interactions in the WT simulations. In the variant simulations, the positively charged guanidinium side chain of Arg697 occasionally forms hydrogen bonds with nearby residues, such as Ser722 and Asn719. However, similar to Trp697 in the WT, Arg697 does not form any long-lasting interactions and thus does not induce any negative structural effects in the simulations.
c.2522T>C	V841A			Uncertain		1	6-33443074-T-C	3	1.86e-6	-8.152	Likely Pathogenic	0.901	Likely Pathogenic	Ambiguous	0.183	Likely Benign										-2.13	Neutral	0.992	Probably Damaging	0.989	Probably Damaging	2.57	Benign	0.02	Affected	3.77	5	0	0	-2.4	-28.05
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.2560C>T	R854C	Likely Benign		Uncertain		1	6-33443112-C-T	3	1.86e-6	-5.082	Likely Benign	0.170	Likely Benign	Likely Benign	0.174	Likely Benign										-2.48	Neutral	1.000	Probably Damaging	0.947	Probably Damaging	4.05	Benign	0.01	Affected	3.88	3	-3	-4	7.0	-53.05
c.2561G>A	R854H	Likely Benign		Uncertain		1	6-33443113-G-A	4	2.48e-6	-3.686	Likely Benign	0.094	Likely Benign	Likely Benign	0.183	Likely Benign										-1.38	Neutral	0.997	Probably Damaging	0.899	Possibly Damaging	4.07	Benign	0.04	Affected	3.88	3	2	0	1.3	-19.05
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.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.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.2578G>A	V860I	Likely Benign		Benign		1	6-33443130-G-A	21	1.30e-5	-4.516	Likely Benign	0.095	Likely Benign	Likely Benign	0.039	Likely Benign										-0.42	Neutral	0.009	Benign	0.006	Benign	4.24	Benign	0.00	Affected	3.77	5	4	3	0.3	14.03
c.2582C>T	S861L	Likely Benign		Uncertain		1	6-33443134-C-T	2	1.24e-6	-4.966	Likely Benign	0.219	Likely Benign	Likely Benign	0.144	Likely Benign										-2.10	Neutral	0.904	Possibly Damaging	0.355	Benign	3.93	Benign	0.07	Tolerated	4.32	3	-3	-2	4.6	26.08
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.2143C>T	P715S (3D Viewer)		GAP	Likely Pathogenic		1	6-33441608-C-T	1	6.20e-7	-7.635	In-Between	0.787	Likely Pathogenic	Ambiguous	0.277	Likely Benign	3.54	Destabilizing	0.0	0.81	Ambiguous	2.18	Destabilizing	0.94	Ambiguous	-7.17	Deleterious	1.000	Probably Damaging	0.998	Probably Damaging	3.43	Benign	0.01	Affected	3.50	9	1	-1	0.8	-10.04	231.8	-14.0	-0.1	0.0	-0.8	0.1							X	Uncertain	Pro715, along with Gly712 and Pro713, are located in a hinge region of an α-helix making a ~90-degree turn (res. Lys705-Leu725). In the WT simulations, the pyrrolidine side chain of Pro715, lacking the backbone amide groups altogether, forces the tight helix turn to take place while also hydrophobically packing with nearby residues (e.g., Leu700, Leu708, Leu714, and Leu718). Leu715, with a normal amide backbone, could potentially affect protein folding and turn formation, although this was not observed in the variant simulations. Additionally, the hydroxyl group of the Ser715 side chain can form hydrogen bonds with the backbone carbonyl group of Gly712 and disrupt the hydrophobic packing arrangement of the leucine residues from the neighboring α-helices, impacting the GAP domain tertiary assembly.
c.2147G>A	R716Q (3D Viewer)		GAP	Conflicting		2	6-33441612-G-A	4	2.48e-6	-8.338	Likely Pathogenic	0.308	Likely Benign	Likely Benign	0.210	Likely Benign	-0.01	Likely Benign	0.0	0.47	Likely Benign	0.23	Likely Benign	0.58	Ambiguous	-3.14	Deleterious	1.000	Probably Damaging	0.990	Probably Damaging	3.35	Benign	0.02	Affected	3.50	9	1	1	1.0	-28.06	250.0	48.9	0.0	0.0	-0.5	0.0						X		Uncertain	The guanidinium group of Arg716, located on the outer surface of an α-helix (res. Leu714-Arg726), forms a salt bridge with the carboxylate group of Asp720. In the variant simulations, the carboxamide group of Gln716 also forms a hydrogen bond with the carboxylate group of Asp720, although this bond is weaker than the Arg716 salt bridge in the WT. Overall, no adverse effects on the protein structure are observed in the simulations. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2162T>G	I721S (3D Viewer)	Likely Pathogenic	GAP	Uncertain		1				-14.032	Likely Pathogenic	0.996	Likely Pathogenic	Likely Pathogenic	0.466	Likely Benign	3.91	Destabilizing	0.1	3.96	Destabilizing	3.94	Destabilizing	2.28	Destabilizing	-5.26	Deleterious	1.000	Probably Damaging	1.000	Probably Damaging	2.21	Pathogenic	0.00	Affected	3.50	9	-1	-2	-5.3	-26.08	203.3	49.3	-0.1	0.0	-1.1	0.0							X	Uncertain	The sec-butyl side chain of Ile721, located on an α-helix (res. Leu714-Arg726), engages in hydrophobic packing with other residues in the hydrophobic inter-helix space, such as Phe420, Tyr417, His693, and Leu717. In the variant simulations, the hydroxyl side chain of Ser721 forms hydrogen bonds with nearby residues, such as Leu717 and His693. Although no major structural changes are observed during the variant simulations, the hydrophilic residue Ser721 could disrupt the hydrophobic packing during folding. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.2596G>A	V866I	Likely Benign		Conflicting		3	6-33443148-G-A	5	3.10e-6	-4.652	Likely Benign	0.118	Likely Benign	Likely Benign	0.059	Likely Benign										-0.39	Neutral	0.957	Probably Damaging	0.541	Possibly Damaging	2.69	Benign	0.27	Tolerated	3.82	4	4	3	0.3	14.03
c.2596G>T	V866L	Likely Benign		Uncertain		1	6-33443148-G-T	1	6.20e-7	-3.352	Likely Benign	0.148	Likely Benign	Likely Benign	0.046	Likely Benign										-0.97	Neutral	0.217	Benign	0.229	Benign	2.71	Benign	0.21	Tolerated	3.82	4	2	1	-0.4	14.03
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.2619C>G	S873R			Uncertain		1	6-33443171-C-G	1	6.20e-7	-5.856	Likely Benign	0.976	Likely Pathogenic	Likely Pathogenic	0.192	Likely Benign										-2.74	Deleterious	0.997	Probably Damaging	0.995	Probably Damaging	2.67	Benign	0.06	Tolerated	3.77	5	0	-1	-3.7	69.11
c.2623G>A	A875T	Likely Benign		Uncertain		1	6-33443175-G-A	1	6.20e-7	-3.793	Likely Benign	0.179	Likely Benign	Likely Benign	0.110	Likely Benign										-1.56	Neutral	0.972	Probably Damaging	0.864	Possibly Damaging	2.72	Benign	0.26	Tolerated	3.77	5	0	1	-2.5	30.03
c.2168C>T	T723I (3D Viewer)	Likely Benign	GAP	Likely Benign		1	6-33441633-C-T	2	1.24e-6	-2.591	Likely Benign	0.120	Likely Benign	Likely Benign	0.045	Likely Benign	-0.39	Likely Benign	0.0	-0.20	Likely Benign	-0.30	Likely Benign	0.26	Likely Benign	-2.09	Neutral	0.088	Benign	0.030	Benign	3.39	Benign	0.03	Affected	3.50	8	0	-1	5.2	12.05	252.3	-31.6	0.0	0.0	-0.2	0.2						X		Uncertain	The hydroxyl group of Thr723, located on the outer surface of an α-helix (res. Leu714-Arg726), continuously forms hydrogen bonds with the backbone carbonyl of Asn719 in the WT simulations, potentially lowering the stability of the α-helix. In the variant simulations, the sec-butyl side chain of Ile723 cannot form any hydrogen bonds, which, in theory, could increase the helix stability. However, because the model ends abruptly at the C-terminus, no definite conclusions can be drawn based on the simulations.
c.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.2635_2636delinsAA	A879K	Likely Benign		Likely Benign		1				-5.877	Likely Benign	0.757	Likely Pathogenic	Likely Benign												-0.71	Neutral	0.969	Probably Damaging	0.593	Possibly Damaging	2.69	Benign	0.21	Tolerated	3.77	5	-1	-1	-5.7	57.10
c.263T>C	V88A	Likely Benign		Uncertain		1				-5.860	Likely Benign	0.993	Likely Pathogenic	Likely Pathogenic	0.050	Likely Benign										-1.22	Neutral	0.053	Benign	0.008	Benign	3.75	Benign	0.00	Affected	4.32	1	0	0	-2.4	-28.05
c.2650C>T	R884W	Likely Benign		Uncertain		1	6-33443202-C-T	5	3.10e-6	-3.785	Likely Benign	0.332	Likely Benign	Likely Benign	0.151	Likely Benign										0.26	Neutral	0.995	Probably Damaging	0.812	Possibly Damaging	2.56	Benign	0.05	Affected	4.32	4	-3	2	3.6	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.2657C>T	A886V	Likely Benign		Uncertain		1	6-33443209-C-T	18	1.12e-5	-4.478	Likely Benign	0.078	Likely Benign	Likely Benign	0.061	Likely Benign										-0.20	Neutral	0.888	Possibly Damaging	0.314	Benign	2.17	Pathogenic	0.00	Affected	4.32	4	0	0	2.4	28.05
c.2668C>T	R890C			Benign		1	6-33443220-C-T	9	5.58e-6	-5.786	Likely Benign	0.402	Ambiguous	Likely Benign	0.200	Likely Benign										-3.38	Deleterious	1.000	Probably Damaging	0.971	Probably Damaging	3.94	Benign	0.04	Affected	4.32	4	-4	-3	7.0	-53.05
c.2669G>A	R890H	Likely Benign		Benign		1	6-33443221-G-A	19	1.18e-5	-3.600	Likely Benign	0.198	Likely Benign	Likely Benign	0.056	Likely Benign										-1.29	Neutral	0.254	Benign	0.134	Benign	3.97	Benign	0.15	Tolerated	4.32	4	2	0	1.3	-19.05
c.2681G>A	G894E	Likely Benign		Uncertain		1	6-33443233-G-A	6	3.72e-6	-5.377	Likely Benign	0.859	Likely Pathogenic	Ambiguous	0.180	Likely Benign										-2.07	Neutral	1.000	Probably Damaging	1.000	Probably Damaging	2.68	Benign	0.01	Affected	4.32	4	0	-2	-3.1	72.06
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.2690C>T	S897L	Likely Benign		Uncertain		1				-4.034	Likely Benign	0.299	Likely Benign	Likely Benign	0.028	Likely Benign										-1.71	Neutral	0.901	Possibly Damaging	0.636	Possibly Damaging	2.66	Benign	0.01	Affected			-3	-2	4.6	26.08
c.2695A>G	I899V	Likely Benign		Benign		1	6-33443247-A-G	6	3.72e-6	-2.569	Likely Benign	0.074	Likely Benign	Likely Benign	0.040	Likely Benign										0.09	Neutral	0.220	Benign	0.078	Benign	2.75	Benign	0.92	Tolerated	4.32	4	4	3	-0.3	-14.03
c.2699C>T	T900M	Likely Benign		Conflicting		2	6-33443251-C-T	14	8.68e-6	-3.852	Likely Benign	0.176	Likely Benign	Likely Benign	0.015	Likely Benign										-0.81	Neutral	0.060	Benign	0.016	Benign	2.79	Benign	0.08	Tolerated	4.32	4	-1	-1	2.6	30.09
c.269T>A	V90E	Likely Benign		Uncertain		1				-4.079	Likely Benign	0.703	Likely Pathogenic	Likely Benign	0.108	Likely Benign										-0.38	Neutral	0.001	Benign	0.000	Benign	4.00	Benign	0.00	Affected	4.32	1	-2	-2	-7.7	29.98
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.2711T>C	M904T	Likely Benign		Uncertain		1				-2.721	Likely Benign	0.668	Likely Pathogenic	Likely Benign	0.042	Likely Benign										-1.15	Neutral	0.277	Benign	0.103	Benign	2.78	Benign	0.18	Tolerated	3.77	5	-1	-1	-2.6	-30.09
c.2713C>T	R905C			Conflicting		2	6-33443265-C-T	15	9.31e-6	-5.578	Likely Benign	0.723	Likely Pathogenic	Likely Benign	0.194	Likely Benign										-3.14	Deleterious	1.000	Probably Damaging	0.980	Probably Damaging	2.57	Benign	0.01	Affected	3.77	5	-4	-3	7.0	-53.05
c.2714G>A	R905H	Likely Benign		Uncertain		1	6-33443266-G-A	8	4.96e-6	-4.182	Likely Benign	0.457	Ambiguous	Likely Benign	0.192	Likely Benign										-1.11	Neutral	1.000	Probably Damaging	0.991	Probably Damaging	2.59	Benign	0.09	Tolerated	3.77	5	2	0	1.3	-19.05
c.2719A>T	S907C	Likely Benign		Likely Benign		1				-6.685	Likely Benign	0.298	Likely Benign	Likely Benign	0.113	Likely Benign										-2.34	Neutral	0.999	Probably Damaging	0.988	Probably Damaging	2.60	Benign	0.02	Affected	3.77	5	0	-1	3.3	16.06
c.2724G>C	Q908H	Likely Benign		Conflicting		4	6-33443276-G-C	1	6.20e-7	-4.658	Likely Benign	0.311	Likely Benign	Likely Benign	0.112	Likely Benign										-0.74	Neutral	0.996	Probably Damaging	0.995	Probably Damaging	2.58	Benign	0.05	Affected	3.77	5	3	0	0.3	9.01
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.272A>G	E91G	Likely Benign		Likely Benign		1				-3.226	Likely Benign	0.783	Likely Pathogenic	Likely Benign	0.110	Likely Benign										-2.18	Neutral	0.947	Possibly Damaging	0.727	Possibly Damaging	3.86	Benign	0.00	Affected	4.32	1	0	-2	3.1	-72.06
c.2735C>A	T912N	Likely Benign		Uncertain		1				-4.260	Likely Benign	0.190	Likely Benign	Likely Benign	0.116	Likely Benign										-1.15	Neutral	0.999	Probably Damaging	0.977	Probably Damaging	3.96	Benign	0.00	Affected	3.77	5	0	0	-2.8	13.00
c.2741A>T	D914V	Likely Benign		Uncertain		1				-4.260	Likely Benign	0.723	Likely Pathogenic	Likely Benign	0.187	Likely Benign										-2.24	Neutral	0.999	Probably Damaging	0.986	Probably Damaging	2.64	Benign	0.01	Affected	3.77	5	-3	-2	7.7	-15.96
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.2750C>G	P917R	Likely Benign		Uncertain		1	6-33443302-C-G	5	3.10e-6	-4.475	Likely Benign	0.363	Ambiguous	Likely Benign	0.142	Likely Benign										-1.70	Neutral	0.642	Possibly Damaging	0.316	Benign	2.68	Benign	0.00	Affected	3.77	5	-2	0	-2.9	59.07
c.2752G>A	A918T	Likely Benign		Uncertain		1	6-33443304-G-A	1	6.20e-7	-4.139	Likely Benign	0.083	Likely Benign	Likely Benign	0.065	Likely Benign										-1.09	Neutral	0.980	Probably Damaging	0.721	Possibly Damaging	2.64	Benign	0.03	Affected	4.32	4	0	1	-2.5	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.2768T>A	I923N	Likely Benign		Uncertain		1				-0.733	Likely Benign	0.712	Likely Pathogenic	Likely Benign	0.108	Likely Benign										-1.16	Neutral	0.991	Probably Damaging	0.793	Possibly Damaging	2.70	Benign	0.13	Tolerated	3.77	5	-2	-3	-8.0	0.94
c.277C>G	R93G	Likely Benign		Uncertain		1				-2.674	Likely Benign	0.400	Ambiguous	Likely Benign	0.093	Likely Benign										-1.69	Neutral	0.103	Benign	0.019	Benign	3.99	Benign	0.00	Affected	4.32	1	-2	-3	4.1	-99.14
c.2809G>C	D937H	Likely Benign		Uncertain		1				-0.733	Likely Benign	0.677	Likely Pathogenic	Likely Benign	0.150	Likely Benign										-1.74	Neutral	1.000	Probably Damaging	0.975	Probably Damaging	2.68	Benign	0.13	Tolerated	3.77	5	-1	1	0.3	22.05
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.2812G>A	G938R	Likely Benign		Uncertain		1				-5.271	Likely Benign	0.732	Likely Pathogenic	Likely Benign	0.141	Likely Benign										-1.11	Neutral	0.999	Probably Damaging	0.985	Probably Damaging	2.74	Benign	0.36	Tolerated	3.77	5	-3	-2	-4.1	99.14
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.2818G>C	G940R	Likely Benign		Benign		1	6-33443370-G-C	5	3.10e-6	-6.169	Likely Benign	0.480	Ambiguous	Likely Benign	0.060	Likely Benign										0.02	Neutral	0.922	Possibly Damaging	0.543	Possibly Damaging	2.73	Benign	0.15	Tolerated	3.77	5	-3	-2	-4.1	99.14
c.2825C>T	P942L	Likely Benign		Uncertain		1	6-33443377-C-T	4	2.48e-6	-5.063	Likely Benign	0.086	Likely Benign	Likely Benign	0.048	Likely Benign										-2.00	Neutral	0.411	Benign	0.239	Benign	2.37	Pathogenic	0.00	Affected	4.32	4	-3	-3	5.4	16.04
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.2835T>A	H945Q	Likely Benign		Conflicting		2	6-33443387-T-A	3	1.86e-6	-5.248	Likely Benign	0.091	Likely Benign	Likely Benign	0.343	Likely Benign										-0.36	Neutral	0.995	Probably Damaging	0.939	Probably Damaging	5.03	Benign	0.06	Tolerated	4.32	4	3	0	-0.3	-9.01
c.2837G>A	G946E	Likely Benign		Benign		3	6-33443389-G-A	13	8.05e-6	-8.793	Likely Pathogenic	0.257	Likely Benign	Likely Benign	0.341	Likely Benign										-0.51	Neutral	0.818	Possibly Damaging	0.355	Benign	4.58	Benign	0.00	Affected	4.32	4	0	-2	-3.1	72.06
c.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.2852A>G	H951R	Likely Benign		Likely Pathogenic		1				-4.964	Likely Benign	0.125	Likely Benign	Likely Benign	0.185	Likely Benign										-1.08	Neutral	0.048	Benign	0.029	Benign	5.46	Benign	0.24	Tolerated	3.77	5	2	0	-1.3	19.05
c.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.2855G>T	G952V	Likely Benign		Uncertain		1				-7.074	In-Between	0.078	Likely Benign	Likely Benign	0.231	Likely Benign										-0.33	Neutral	0.000	Benign	0.000	Benign	3.20	Benign	0.02	Affected	3.77	5	-1	-3	4.6	42.08
c.2858C>A	P953Q	Likely Benign		Uncertain		1				-6.038	Likely Benign	0.079	Likely Benign	Likely Benign	0.086	Likely Benign										-0.78	Neutral	0.058	Benign	0.015	Benign	2.78	Benign	0.29	Tolerated	3.77	5	0	-1	-1.9	31.01
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.2864C>T	S955F			Conflicting		4	6-33443416-C-T	95	5.89e-5	-7.374	In-Between	0.176	Likely Benign	Likely Benign	0.093	Likely Benign										-1.73	Neutral	0.977	Probably Damaging	0.721	Possibly Damaging	2.32	Pathogenic	0.00	Affected	3.77	5	-3	-2	3.6	60.10
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.2873A>C	H958P	Likely Benign		Benign		1	6-33443425-A-C	2	1.24e-6	-8.369	Likely Pathogenic	0.068	Likely Benign	Likely Benign	0.204	Likely Benign										-0.36	Neutral	0.925	Possibly Damaging	0.316	Benign	4.14	Benign	0.10	Tolerated	3.77	5	0	-2	1.6	-40.02
c.2881C>T	H961Y	Likely Benign		Conflicting		2	6-33443433-C-T	3	1.86e-6	-8.051	Likely Pathogenic	0.157	Likely Benign	Likely Benign	0.102	Likely Benign										-1.07	Neutral	0.716	Possibly Damaging	0.147	Benign	4.10	Benign	0.55	Tolerated	3.77	5	0	2	1.9	26.03
c.2888A>G	H963R	Likely Benign		Uncertain		1	6-33443440-A-G	8	4.96e-6	-8.952	Likely Pathogenic	0.169	Likely Benign	Likely Benign	0.081	Likely Benign										-1.28	Neutral	0.001	Benign	0.003	Benign	4.15	Benign	0.24	Tolerated	3.77	5	2	0	-1.3	19.05
c.28C>T	R10W	Likely Benign		Uncertain		1	6-33420292-C-T	2	1.30e-6	-5.707	Likely Benign	0.503	Ambiguous	Likely Benign	0.236	Likely Benign										-0.31	Neutral	0.964	Probably Damaging	0.190	Benign	4.10	Benign	0.00	Affected	4.32	1	2	-3	3.6	30.03
c.2900G>T	R967L	Likely Benign		Uncertain		1	6-33443452-G-T	1	6.20e-7	-3.496	Likely Benign	0.164	Likely Benign	Likely Benign	0.123	Likely Benign										-0.99	Neutral	0.959	Probably Damaging	0.586	Possibly Damaging	4.15	Benign	0.75	Tolerated	4.32	2	-2	-3	8.3	-43.03
c.2909A>G	E970G	Likely Benign		Benign		1				-0.167	Likely Benign	0.139	Likely Benign	Likely Benign	0.139	Likely Benign										-0.93	Neutral	0.144	Benign	0.058	Benign	4.09	Benign	0.10	Tolerated	4.32	2	0	-2	3.1	-72.06
c.2912C>A	P971H	Likely Benign		Uncertain		1	6-33443464-C-A	1	6.20e-7	-5.243	Likely Benign	0.086	Likely Benign	Likely Benign	0.039	Likely Benign										-1.11	Neutral	0.898	Possibly Damaging	0.477	Possibly Damaging	3.89	Benign	0.00	Affected	4.32	2	-2	0	-1.6	40.02
c.2914C>G	P972A	Likely Benign		Uncertain		1	6-33443466-C-G	1	6.20e-7	-0.167	Likely Benign	0.045	Likely Benign	Likely Benign	0.046	Likely Benign										-0.89	Neutral	0.016	Benign	0.011	Benign	4.29	Benign	0.07	Tolerated	4.32	2	-1	1	3.4	-26.04
c.2914C>T	P972S	Likely Benign		Uncertain		1	6-33443466-C-T	4	2.48e-6	-4.008	Likely Benign	0.058	Likely Benign	Likely Benign	0.074	Likely Benign										-0.38	Neutral	0.001	Benign	0.002	Benign	4.28	Benign	0.05	Affected	4.32	2	-1	1	0.8	-10.04
c.2924C>A	T975N	Likely Benign		Uncertain		1	6-33443476-C-A	1	6.20e-7	-4.671	Likely Benign	0.089	Likely Benign	Likely Benign	0.100	Likely Benign										-0.58	Neutral	0.586	Possibly Damaging	0.302	Benign	4.13	Benign	0.07	Tolerated	4.32	2	0	0	-2.8	13.00
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.2924C>T	T975I	Likely Benign		Uncertain		1	6-33443476-C-T	6	3.72e-6	-3.912	Likely Benign	0.164	Likely Benign	Likely Benign	0.068	Likely Benign										-1.66	Neutral	0.411	Benign	0.239	Benign	4.11	Benign	0.66	Tolerated	4.32	2	0	-1	5.2	12.05
c.2928T>G	F976L	Likely Benign		Uncertain		1				-2.432	Likely Benign	0.825	Likely Pathogenic	Ambiguous	0.212	Likely Benign										-0.87	Neutral	0.264	Benign	0.102	Benign	4.20	Benign	0.53	Tolerated	4.32	2	2	0	1.0	-34.02
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.2935T>C	F979L	Likely Benign		Uncertain		1				-2.341	Likely Benign	0.870	Likely Pathogenic	Ambiguous	0.228	Likely Benign										-1.00	Neutral	0.625	Possibly Damaging	0.430	Benign	4.22	Benign	0.73	Tolerated	4.32	2	2	0	1.0	-34.02
c.2945A>G	Y982C	Likely Benign		Likely Benign		1	6-33443497-A-G	2	1.24e-6	-6.256	Likely Benign	0.746	Likely Pathogenic	Likely Benign	0.195	Likely Benign										-1.67	Neutral	0.997	Probably Damaging	0.923	Probably Damaging	3.87	Benign	0.00	Affected	4.32	1	0	-2	3.8	-60.04
c.2948G>A	S983N			Likely Benign		1	6-33443500-G-A	6	3.72e-6	-5.604	Likely Benign	0.909	Likely Pathogenic	Ambiguous	0.136	Likely Benign										-1.78	Neutral	0.991	Probably Damaging	0.988	Probably Damaging	2.04	Pathogenic	0.00	Affected	4.32	1	1	1	-2.7	27.03
c.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.2962C>T	L988F	Likely Benign		Uncertain		1	6-33443514-C-T	1	6.20e-7	-4.368	Likely Benign	0.356	Ambiguous	Likely Benign	0.135	Likely Benign										-1.70	Neutral	0.977	Probably Damaging	0.900	Possibly Damaging	2.69	Benign	0.00	Affected	4.32	2	2	0	-1.0	34.02
c.2971G>A	G991R	Likely Benign		Conflicting		3	6-33443523-G-A	8	4.96e-6	-3.934	Likely Benign	0.411	Ambiguous	Likely Benign	0.102	Likely Benign										-1.20	Neutral	0.984	Probably Damaging	0.772	Possibly Damaging	4.11	Benign	0.01	Affected	4.32	2	-3	-2	-4.1	99.14
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.2987C>G	P996R	Likely Benign		Benign		1				-4.457	Likely Benign	0.141	Likely Benign	Likely Benign	0.040	Likely Benign										-1.04	Neutral	0.144	Benign	0.085	Benign	4.26	Benign	0.01	Affected	4.32	4	-2	0	-2.9	59.07
c.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.2998A>G	I1000V	Likely Benign		Uncertain		2				-4.102	Likely Benign	0.098	Likely Benign	Likely Benign	0.086	Likely Benign										-0.20	Neutral	0.437	Benign	0.170	Benign	2.76	Benign	0.81	Tolerated	4.32	4	3	4	-0.3	-14.03
c.3002T>C	L1001P	Likely Benign		Uncertain		1				-3.071	Likely Benign	0.209	Likely Benign	Likely Benign	0.113	Likely Benign										-1.02	Neutral	0.966	Probably Damaging	0.690	Possibly Damaging	2.65	Benign	0.00	Affected	4.32	4	-3	-3	-5.4	-16.04
c.3009C>G	S1003R			Uncertain		1				-5.113	Likely Benign	0.991	Likely Pathogenic	Likely Pathogenic	0.141	Likely Benign										-1.88	Neutral	0.999	Probably Damaging	0.996	Probably Damaging	2.48	Pathogenic	0.00	Affected	3.77	5	0	-1	-3.7	69.11
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.3022G>A	D1008N	Likely Benign		Likely Benign		1	6-33443574-G-A	3	1.86e-6	-4.045	Likely Benign	0.714	Likely Pathogenic	Likely Benign	0.128	Likely Benign										-2.15	Neutral	0.999	Probably Damaging	0.997	Probably Damaging	2.75	Benign	0.01	Affected	3.77	5	2	1	0.0	-0.98
c.3023A>G	D1008G			Uncertain		1	6-33443575-A-G	1	6.20e-7	-3.213	Likely Benign	0.742	Likely Pathogenic	Likely Benign	0.203	Likely Benign										-2.84	Deleterious	0.999	Probably Damaging	0.997	Probably Damaging	2.65	Benign	0.01	Affected	3.77	5	-1	1	3.1	-58.04
c.3026A>C	E1009A	Likely Pathogenic		Uncertain		1				-3.118	Likely Benign	0.679	Likely Pathogenic	Likely Benign	0.109	Likely Benign										-3.06	Deleterious	0.980	Probably Damaging	0.630	Possibly Damaging	2.39	Pathogenic	0.01	Affected	3.77	5	0	-1	5.3	-58.04
c.3038C>G	S1013C	Likely Benign		Uncertain		1	6-33443590-C-G	4	2.48e-6	-6.745	Likely Benign	0.110	Likely Benign	Likely Benign	0.058	Likely Benign										-2.06	Neutral	0.898	Possibly Damaging	0.579	Possibly Damaging	2.64	Benign	0.05	Affected	3.77	5	0	-1	3.3	16.06
c.303C>A	H101Q	Likely Benign		Uncertain		1	6-33432168-C-A	1	6.20e-7	-2.827	Likely Benign	0.124	Likely Benign	Likely Benign	0.147	Likely Benign										-0.37	Neutral	0.824	Possibly Damaging	0.880	Possibly Damaging	4.24	Benign	0.00	Affected	4.32	1	3	0	-0.3	-9.01
c.3041G>T	G1014V	Likely Benign		Uncertain		1				-4.612	Likely Benign	0.181	Likely Benign	Likely Benign	0.053	Likely Benign										-2.47	Neutral	0.818	Possibly Damaging	0.377	Benign	2.72	Benign	0.06	Tolerated	3.77	5	-1	-3	4.6	42.08
c.3048C>A	D1016E	Likely Benign		Likely Benign		1	6-33443600-C-A	2	1.24e-6	-3.422	Likely Benign	0.216	Likely Benign	Likely Benign	0.017	Likely Benign										-0.37	Neutral	0.008	Benign	0.028	Benign	2.64	Benign	0.65	Tolerated	3.77	5	2	3	0.0	14.03