Table 2 Summary of computational analysis of rare/novel SNVs

 1144

P289S^a

Proline (P; non-polar side chain) > serine (S; polar uncharged side chain)

0.8282

3.49

1.000

• Giacopuzzi et al. (2018) [19] REVEL: 0.901

• VEST3: 0.951

• iFISH: 0.9866

• MutationAssessor: 4.425 (high)

• SIFT: 0

• Tightly packed side chain buried in the same hydrophobic region as M221, in the breach at the top of the A-sheet, and the beginning of the RCL

• Serine is not tolerated sterically as it is larger – likely causing disruption to role of this strategic portion of the protein

 4668

I50N

Isoleucine (I; hydrophobic side chain) > asparagine (N; polar uncharged side chain)

0.8153

2.69

1.000

Giacopuzzi et al. (2018) [19]

• REVEL: 0.873

• VEST3: 0.706

• iFISH: 0.9825

• MutationAssessor: 4.41 (high)

• SIFT: 0

• Highly conserved residue

• Polar side chain introduced to a very hydrophobic core of the protein

• Will destabilize hydrophobic core

 12,642

D341V

Aspartic acid (D; negatively charged side chain)

>valine (V; hydrophobic side chain)

0.8651

0.99

0.998

Giacopuzzi et al. (2018) [19]

• REVEL: 0.599

• VEST3: 0.765

• iFISH: 0.9823

• MutationAssessor: 4.06 (high)

• SIFT: 0.001

• Conserved residue

• Borderline significant change in protein stability

• In a buried location found at the “Breach” region of the protein at the base of the RCL loop

• Change to valine would eliminate aspartic acid hydrogen bonding to adjacent K343 and possibly affect RCL conformation

 14,271

M221 T

Methionine (M; hydrophobic side chain)

>threonine (T; polar uncharged side chain)

0.8186

2.93

0.997

Giacopuzzi et al. (2018) [19]

• REVEL: 0.933

• VEST3: 0.778

• iFISH: 0.9826

• MutationAssessor: 4.74 (high)

• SIFT: 0.001

• Highly conserved residue

• Tightly packed side chain buried in hydrophobic region in the breach at top of α-sheet and beginning of RCL

• Threonine would be sterically tolerated due to smaller size but would not have same impact as Methionine on tight packing in strategic area of protein

 15,230

V210E

Valine (V; hydrophobic side chain)

>glutamic acid (E; negatively charged side chain)

0.7162

1.37

0.818

Giacopuzzi et al. (2018) [19]

• REVEL: 0.752

• VEST3: 0.618

• iFISH: 0.9338

• MutationAssessor: 3.745 (high)

• SIFT: 0.002

• Not a highly conserved residue

• Residue participates in tight hydrophobic packing near the tip of a β–strand hairpin

• Introduction of glutamic acid could cause charge repulsion with D211 and disrupt packing of β-hairpin or could introduce a new h-bond with nearby N390

 4293†& 5564†

P28L^a

Proline (P; non-polar side chain)

>leucine (L; hydrophobic side chain)

0.8205

1.17

0.648

Giacopuzzi et al. (2018) [19]

• REVEL: 0.387

• VEST3: 0.404

• iFISH: 0.7976

• MutationAssessor: 2.86 (medium)

• SIFT: 0.038

• Highly conserved residue

• P28 is near the N-terminus and the side chain packs against P23.

• Change to the larger hydrophobic leucine would be sterically permissible as the side chain is surface-accessible.

• Possible that the wildtype Proline is necessary to kink the helix for the tight packing to occur, and the conformation of N-terminal helix interaction with the rest of the protein

 21,034

P369H

Proline (P; non-polar side chain)

>histidine (H; positively charged side chain)

0.8784

3.36

1.000

Giacopuzzi et al. (2018) [19]

• REVEL: 0.834

• VEST3: 0.945

• iFISH: 0.9877

• MutationAssessor: 4.755 (high)

• SIFT: 0

• Buried location found at end of the RCL loop

• Change to histidine would disrupt packing and affect RCL conformation

 24,319

A142D

Alanine (A; small hydrophobic side chain)

>aspartic acid (D; negatively charged side chain)

0.7958

1.03

0.992

Giacopuzzi et al. (2018) [19]

• REVEL: 0.615

• VEST3: 0.694

• iFISH: 0.9591

• MutationAssessor: 3.51 (high)

• SIFT: 0.003

Silva et al., (2016) [20]

• PolyPhen-2: 0.99

• Highly conserved residue

• Change to aspartic acid could be sterically problematic as larger charged side chain is introduced to a hydrophobic pocket and could destabilize it

 9533

M385 T

Methionine (M; hydrophobic side chain)

>threonine (T; polar uncharged side chain)

0.8722

3.34

0.134

Giacopuzzi et al. (2018) [19]

• REVEL: 0.668

• VEST3: 0.738

• iFISH: 0.801

• MutationAssessor: 1.97 (medium)

• SIFT: 0.094

• Conserved residue

• Residue in buried core of protein and makes at least 12 hydrophobic contacts in the core

• Change to threonine would shorten the side chain and disrupt core packing; note the significant stability drop

 21,636

V333 M

Valine (V; hydrophobic side chain)

>methionine (M; hydrophobic side chain)

0.7237

−0.25

0.990

Giacopuzzi et al. (2018) [19]

• REVEL: 0.539

• VEST3: 0.676

• iFISH: 0.8378

• MutationAssessor: 1.985 (medium)

• SIFT: 0.079

Silva et al., (2016) [20]

• PolyPhen-2: 0.53

• Buried location with low ASA; found within the beta-sheet region

• Larger/longer side-chain

• Methionine may sterically clash in the buried location

 2343

I9N [includes precursor]^a

Isoleucine (I; hydrophobic side chain) >

asparagine (N; polar uncharged side chain)

0.3387

N/A

0.517

Giacopuzzi et al. (2018) [19]

• REVEL: 0.453

• VEST3: 0.291

• iFISH: 0.3779

• MutationAssessor: 1.1 (low)

• SIFT: 0.001

• Not included in Fig. 1 visualization (no structural information on this portion of the protein)

 10,889

Q40R^a

Glutamine (Q; polar uncharged side chain)

>arginine (R; positively charged side chain)

0.6589

−0.35

0.018

Giacopuzzi et al. (2018) [19]

• REVEL: 0.311

• VEST3: 0.092

• iFISH: 0.5284

• MutationAssessor: 1.515 (low)

• SIFT: 0.24

• Conserved residue

• Change to the larger Arginine side chain would present steric problems, despite its accessibility

• Q40 hydrogen bonds to V302 and while the larger side chain could also hydrogen bond, it could disrupt packing of the helix that holds V302

 17,657

K174E^a

Lysine (K; positively charged side chain) >

glutamic acid (E; negatively charged side chain)

0.5053

0.21

0.030

Giacopuzzi et al. (2018) [19]

• REVEL: 0.622

• VEST3: 0.681

• iFISH: 0.6117

• MutationAssessor: 2.24 (medium)

• SIFT: 0.208

• Moderately conserved residue

• Change to side chain is sterically tolerated as a smaller side chain is introduced in to a solvent-accessible loop of the protein

 76,430

H262Y

Histidine (H; positively charged side chain)

>tyrosine (Y; largely hydrophobic side chain, but hydroxyl group can participate in hydrogen bonds or also be phosphorylated)

0.6708

−0.68

0.040

Giacopuzzi et al. (2018) [19]

• REVEL: 0.086

• VEST3: 0.144

• iFISH: 0.5173

• MutationAssessor: 1.54 (low)

• SIFT: 0.042

Silva et al. (2016) [20]

• Polyphen-2: 0.06 (21)

• Highly conserved residue

• Tightly packed side chain buried

• Histidine is involved in 3 hydrogen bonds – to backbone atoms of residues N265, E266, K234

• Tyrosine might not be tolerated sterically because it is larger

 CA97

E204K^a

Glutamic acid (E; negatively charged side chain)

>lysine (K; positively charged side chain)

0.1021

− 0.70

0.000

Giacopuzzi et al. (2018) [19]

• REVEL: 0.457

• VEST3: 0.648

• iFISH: 0.1155

• MutationAssessor: − 0.625 (low)

• SIFT: 0.921

• Not a conserved residue, larger lysine chain is sterically tolerated & can make similar contacts.

• Little change in protein stability is predicted. Although variant could affect RCL from a distance

 23,523

A325P

Alanine (A; small hydrophobic side chain)

>proline (P; non-polar side chain)

0.0878

0.72

0.000

Giacopuzzi et al. (2018) [19]

• REVEL: 0.214

• VEST3: 0.143

• iFISH: 0.4862

• MutationAssessor: 0.265 (medium)

• SIFT: 0.411

• Not conserved

• Insignificant change in protein stability

• In a surface-accessible loop, that could play a role in an alternate trypsin binding site