Background
Methods
Subjects
Laboratory analyses
Computational modeling and variant predictions
Predicting pathogenicity
Benchmarking analysis of SVM predictions
Measurements of binary prediction of SERPINA1 variants
Results
Patient characteristics
Patient ID | AAT serum level (mg/dL) | CRP (mg/dL) | Previous phenotypes | Age (yrs) | Sex | NGS DNA analysis | ||
---|---|---|---|---|---|---|---|---|
Novel mutant nucleotide changes | Consequence | Genotype | ||||||
Splice variants | ||||||||
2250 | 70 | 5.9 | – | 65 | F | Novel splice variant: G > C at position +1 of intron 1C | E376D – M3 allele | |
24023 | 62.8 | – | 71 | M | 917 + 1G > A | Affects the normal mRNA splicing. | E376D – M3 allele | |
Deletions | ||||||||
10724 | 52 | 7.3 | M3 | 49 | M | A347fs: Novel 1 bp deletion (1112delC) at position 347 | Frameshift that extends the protein by 5 amino acids | E376D – M3 allele |
Stop codons | ||||||||
6326e | 2 | 0 | Z/M1 | 57 | F | Q156X: C > T at Chr14:94849037 (GRCh.37.p13) c.538C > T | Insertion of stop codon at position 156 | Q156X E342K – Z allele V213A – M1 allele |
6376e | 98 | 7 | I | 54 | M | Q156X: C > T at Chr14:94849037 (GRCh.37.p13) c.538C > T | Insertion of stop codon at position 156 | Q156X R39C – I allele |
19771 | 91.4 | – | M | 57 | G192Fs: 1 bp deletion at Chr14:94847477 (GRCh37.p13) c.647_647delG | Predicted to produce a premature stop codon at amino acid 214, leading to a premature termination on exon III | ||
Single-nucleotide variations | ||||||||
CA97 | 112 | – | M3 M2/4 | 65 | F | GAG > AAG | E204K – rarea E376D – M3 allele R101H – M2/M4 allele | |
1144 | 72 | – | M1 | 61 | M | CCC > TCC | P289S – rarea V213A – M1 allele | |
2343 | 86 | 3.6 | M1 | 60 | F | ATC > AAC | I9N [includes precursor] – rarea V213A – M1 allele | |
4293d | 66 | 0.5 | M1 | 54 | M | CCC > CTC | Q0Bellingham – insertion of stop codon at position 156 | P28L – rarea K217X – Q0Bellingham V213A – M1 allele |
5564d | 67 | 1.6 | M1 | 51 | F | CCC > CTC | Q0Bellingham – insertion of stop codon at position 156 | P28L – rarea K217X – Q0Bellingham V213A – M1 allele |
4668 | 78 | 2.2 | M3 | 62 | M | ATC > AAC | I50N (PiTijarafe) – rare E376D – M3 allele | |
9533 | 73 | 0.9 | – | 60 | M | ATG > ACG | M385 T – rarec M allele | |
10889 | – | 12.5 | M3 | 34 | F | CAG > CGG | Q40R – rarea E376D – M3 allele | |
12642 | 89 | 0.6 | M3 | 66 | F | GAC > CTC | D341V – rareb E376D – M3 allele P = L118 – no amino acid change | |
14271 | 47 | 0.6 | Z/M1 | 61 | F | ATG > ACG | M221 T – rarea V213A – M1 allele E342K – Z allele | |
15230 | 34 | 1 | Z/M1 | 72 | M | GTG > GAG | V210E (Ncambodia/Pierre-bénite) – rarea V213A – M1 allele E342K – Z allele | |
17,657 | 160 | 1.5 | M3/M4 | 87 | M | AAG > GAG | K174E – NOVELa E376D – M3 allele R101H – M2/M4 allele | |
21034 | 121.2 | – | – | 47 | F | CCC > CAC | P369H – rare E264V – S allele | |
21636 | 88.4 | – | – | 58 | M | GTG > ATG | V333 M – rarec E376D – M3 allele R101H – M4 allele | |
23523 | 118.6 | – | – | 48 | F | GCA > CCA | A325P (Nvestenanova) – rarea R223C – F allele E376D – M3 allele R101H – M4 allele | |
24319 | 79.3 | – | – | 57 | F | GCC > GAC | A142D – rarec E264V – S allele V213A – M1 allele | |
76430 | 74.8 | – | – | 59 | M | CAC > TAC | H262Y – rarec M allele |
SERPINA1 mutations
Splice variants
Base pair deletions
Stop codons
SNVs
Computational analysis of 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 |
Probably deleterious variants
Possibly deleterious variants
Possibly neutral variants
Probably neutral variants
Benchmarking of SVM predictions
SERPINA1 mutation | Species | Sequence Identity (%) | SVM probability | ΔΔG (FoldX) | PolyPhen-2 Score |
---|---|---|---|---|---|
Pathogenic sequence variants (ClinVar) | |||||
P369L |
Homo sapiens
| 100 |
0.8496
|
1.510
|
1.0000
|
P369S | Homo sapiens | 100 |
0.8353
|
4.410
|
1.0000
|
P369T | Homo sapiens | 100 |
0.8599
|
2.810
|
1.0000
|
M358R | Homo sapiens | 100 |
0.7853
| 0.431 | 0.0190 |
E342K | Homo sapiens | 100 |
0.8413
|
2.090
|
1.0000
|
E264V | Homo sapiens | 100 |
0.8619
|
1.660
|
1.0000
|
D256V | Homo sapiens | 100 |
0.8708
|
1.950
|
0.9850
|
G225R | Homo sapiens | 100 |
0.8975
|
5.160
|
0.9820
|
R223C | Homo sapiens | 100 |
0.8954
| −0.350 |
0.9950
|
I92N | Homo sapiens | 100 |
0.8215
|
3.600
|
1.0000
|
G67E | Homo sapiens | 100 |
0.8680
|
26.290
|
1.0000
|
S53F | Homo sapiens | 100 |
0.8687
|
19.860
|
1.0000
|
L41P | Homo sapiens | 100 |
0.7585
|
3.240
|
0.6010
|
R39C | Homo sapiens | 100 |
0.8672
|
2.110
|
1.0000
|
A336T | Homo sapiens | 100 |
0.8479
|
3.450
|
1.0000
|
G115S | Homo sapiens | 100 |
0.7826
|
1.610
|
0.9990
|
F52S | Homo sapiens | 100 |
0.7900
|
6.020
|
1.0000
|
Benign sequence variants (ClinVar) | |||||
E376D | Homo sapiens | N/A | 0.2991 |
1.850
| 0 |
E363K | Homo sapiens | N/A | 0.4172 | −0.900 | 0.11 |
A284S | Homo sapiens | N/A | 0.3445 | −0.240 | 0.139 |
V213A | Homo sapiens | N/A | 0.1161 | −0.100 | 0 |
R101H | Homo sapiens | N/A | 0.0576 | −0.533 | 0 |
Benign sequence variants (Primate neutral variants) | |||||
P21Q | Hylobates sp. ECACC | 95 |
0.363
|
0.72
| 0.014 |
F23L | Papio anubis | 92 | 0.3762 | 0.38 | 0 |
T27A |
Gorilla gorilla
| 98 | 0.4118 |
1.34
| 0 |
N29K | Hylobates sp. ECACC | 95 | 0.4365 | −0.63 | 0.178 |
N29S | Papio anubis | 92 | 0.0839 | 0.13 | 0 |
T48S | Hylobates sp. ECACC | 95 | 0.4991 | 0.51 | 0 |
I50V | Gorilla gorilla | 98 |
0.6687
|
0.96
|
0.767
|
D74S | Chlorocebus sabaeus | 92 | 0.0588 | 0.38 | 0 |
N81H | Pongo abelii | 96 | 0.462 | 0.27 | 0.007 |
I92V | Pongo abelii; Hylobates sp. ECACC | 96; 95 |
0.7931
|
1.06
| 0.006 |
Q105K | Papio anubis | 92 | 0.2178 | −0.5 | 0.001 |
N116S | Gorilla gorilla | 98 |
0.751
|
1.9
| 0.311 |
K136N | Papio anubis | 92 | 0.4292 | 0.07 | 0 |
E141D | Pongo abelii | 96 | 0.4052 | 0.77 | 0.002 |
G148E | Chlorocebus sabaeus | 92 | 0.219 | −0.46 | 0 |
D159N | Papio anubis | 92 | 0.165 | −1.21 | 0 |
Q212E | Pongo abelii | 96 | 0.2568 | 0.35 | 0 |
V213A | Hylobates sp. ECACC | 95 | 0.1161 | −0.1 | 0 |
Q230Y | Papio anubis | 92 | 0.0803 | 0.49 | 0 |
Q230H | Hylobates sp. ECACC | 95 | 0.0545 | 0.41 | 0 |
K233E | Papio anubis | 92 | 0.0787 | 0.58 | 0 |
D270E | Hylobates sp. ECACC | 95 | 0.2543 | −0.03 | 0 |
I271V | Gorilla gorilla | 98 | 0.1015 | 0.52 | 0 |
D280N | Pongo abelii | 96 | 0.1407 |
0.82
| 0 |
S285N | Chlorocebus sabaeus | 92 | 0.0554 | −1.6 | 0 |
S292A | Chlorocebus sabaeus | 92 | 0.3206 | −0.14 | 0.003 |
S301R | Pongo abelii | 96 | 0.1328 | −0.54 | 0.011 |
S301T | Hylobates sp. ECACC | 95 | 0.0724 | 0 | 0 |
S313G | Hylobates sp. ECACC | 95 |
0.606
|
0.63
| 0.05 |
E324D | Chlorocebus sabaeus | 92 | 0.0978 |
0.62
| 0 |
A332V | Homo sapiens | 99 | 0.1255 |
1.88
|
0.99
|
I360V | Gorilla gorilla | 98 | 0.1547 |
0.73
| 0 |
L383H | Hylobates sp. ECACC | 95 |
0.8834
|
3.08
|
1
|
M385I | Papio anubis | 92 |
0.7686
|
3.15
| 0.001 |
M385 V | Pan troglodytes; Pongo abelii | 99; 96 |
0.7566
|
2.44
| 0.001 |
TPR (sensitivity) | TNR (specificity) | PPV | NPV | ACC | BACC | |
---|---|---|---|---|---|---|
SVM | 1.0 | 0.825 | 0.720 | 1.0 | 0.879 | 0.913 |
Polyphen2 | 0.944 | 0.925 | 0.85 | 0.974 | 0.931 | 0.935 |