Table 2.
Summary of computational analysis of rare/novel SNVs
| Patient ID | Novel mutation | Amino acid change | Analysis | ||||
|---|---|---|---|---|---|---|---|
| SVM probability | ΔΔG (FoldX) | PolyPhen-2 Score | Comparison with previous computational characterizations | Comments on side chain structure | |||
| Probably deleterious mutations | |||||||
| 1144 | P289Sa | 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† | P28La |
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 |
| Possibly deleterious mutations | |||||||
| 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 |
| Possibly neutral mutations | |||||||
| 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 | Q40Ra |
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 | K174Ea |
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 |
| Probably neutral mutations | |||||||
| CA97 | E204Ka |
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 |
All sequence variants are reported without the 24 amino acid precursor unless otherwise stated; avariants previously reported in the dbSNP only
Computational analysis (excludes MutationAssessor): numbers in bold, deleterious score; numbers in normal text, neutral score; RCL Reactive center loop, SVM Single vector machine, ASA Accessible surface area