Difference between revisions of "Sequence-based mutation analysis Gaucher Disease"
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− | The aim of this task was to carry out a thorough analysis of ten mutations and to classify them as disease-causing and non-disease causing. The mutations have been selected by another group from our [[Researching SNPs Gaucher Disease|set of |
+ | The aim of this task was to carry out a thorough analysis of ten mutations and to classify them as disease-causing and non-disease causing. The mutations have been selected by another group from our [[Researching SNPs Gaucher Disease|set of mutations]] such that their impact had been unknown for us prior to this task. We investigated the provided mutations with respect to their physicochemical properties, structural features, as well as their conservation and employed the tools, SIFT, Polyphen2, as well as SNAP for predicting their impact on the phenotype. For quantifying to which extend the mutations are disease causing, we assigend a ''disease score'' where -1 means ''non-disease causing'', 0 ''ambiguous'', and 1 ''disease causing''. We averaged the disease scores to obtain a final prediction which we compared with the true impact of the mutation on the phenotype. Technical details are reported in our [[Gaucher_Task06_Protocol|protocol]]. |
== Mutations == |
== Mutations == |
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+ | <xr id="tab:mutations"/> contains five randomly chosen Gaucher disease-causing and five non-disease-causing mutations. Disease causing mutations were sampled from the HGMD whereas non-disease causing mutations were sampled from a set of mutations which were present in the dbSNP but not in the HGMD. Reference sequence was [http://www.uniprot.org/uniprot/p04062 P04062] which has a 39 residue signal peptide. The ten mutations listed in <xr id="tab:mutations"/> were investigated in the following. |
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<figtable id="tab:mutations"> |
<figtable id="tab:mutations"> |
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== Physicochemical properties == |
== Physicochemical properties == |
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+ | We compared the charge, polarity, size, and the aromatic character of the wild-type and mutant amino-acid and assigned a disease-score of 1 to those mutations, which have a severe impact on the physicochemical properties (cf. <xr id="tab:props"/>). Mutations number 3 changes the polarity since glutamate is acidic but lysine basic. We also considered mutation number 5 and 7 disease-causing as tryptophan is aromatic and unpolar, in contrast to the target residues. Substituting alanine, which is small and unpolar, by the long and acidic aspartate might also impact the structure and function of the protein. |
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<figtable id="tab:props"> |
<figtable id="tab:props"> |
Revision as of 13:27, 17 June 2012
The aim of this task was to carry out a thorough analysis of ten mutations and to classify them as disease-causing and non-disease causing. The mutations have been selected by another group from our set of mutations such that their impact had been unknown for us prior to this task. We investigated the provided mutations with respect to their physicochemical properties, structural features, as well as their conservation and employed the tools, SIFT, Polyphen2, as well as SNAP for predicting their impact on the phenotype. For quantifying to which extend the mutations are disease causing, we assigend a disease score where -1 means non-disease causing, 0 ambiguous, and 1 disease causing. We averaged the disease scores to obtain a final prediction which we compared with the true impact of the mutation on the phenotype. Technical details are reported in our protocol.
Contents
Mutations
<xr id="tab:mutations"/> contains five randomly chosen Gaucher disease-causing and five non-disease-causing mutations. Disease causing mutations were sampled from the HGMD whereas non-disease causing mutations were sampled from a set of mutations which were present in the dbSNP but not in the HGMD. Reference sequence was P04062 which has a 39 residue signal peptide. The ten mutations listed in <xr id="tab:mutations"/> were investigated in the following.
<figtable id="tab:mutations">
Nr | Position | From | To |
---|---|---|---|
1 | 99 | H | R |
2 | 211 | V | I |
3 | 150 | E | K |
4 | 236 | L | P |
5 | 248 | W | R |
6 | 509 | L | P |
7 | 351 | W | C |
8 | 423 | A | D |
9 | 482 | D | N |
10 | 83 | R | S |
Randomly selected mutations from HGMD and dbSNP which were used for the sequence-based mutation analysis. </figtable>
Physicochemical properties
We compared the charge, polarity, size, and the aromatic character of the wild-type and mutant amino-acid and assigned a disease-score of 1 to those mutations, which have a severe impact on the physicochemical properties (cf. <xr id="tab:props"/>). Mutations number 3 changes the polarity since glutamate is acidic but lysine basic. We also considered mutation number 5 and 7 disease-causing as tryptophan is aromatic and unpolar, in contrast to the target residues. Substituting alanine, which is small and unpolar, by the long and acidic aspartate might also impact the structure and function of the protein.
<figtable id="tab:props">
Nr | Wildtype | Mutant | Disease score | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
AA | Charge | Polarity | Size | Aromatic | AA | Charge | Polarity | Size | Aromatic | ||
1 | H | negative | polar | large | no | R | negative | polar | large | no | -1 |
2 | V | neutral | unpolar | medium | no | I | neutral | unpolar | medium | no | -1 |
3 | E | positive | polar | large | no | K | negative | polar | large | no | 1 |
4 | L | neutral | unpolar | medium | no | P | neutral | unpolar | medium | no | -1 |
5 | W | neutral | unpolar | large | yes | R | negative | polar | large | no | 1 |
6 | L | neutral | unpolar | medium | no | P | neutral | unpolar | medium | no | -1 |
7 | W | neutral | unpolar | large | yes | C | neutral | polar | small | no | 1 |
8 | A | neutral | unpolar | small | no | D | positive | polar | medium | no | 1 |
9 | D | positive | polar | medium | no | N | neutral | polar | medium | no | -1 |
10 | R | negative | polar | large | no | S | neutral | polar | small | no | 0 |
Physicochemical properiets of the wildtype and mutatant amino acid which were used to classify the mutation as severe or non-severe. </figtable>
Structural analysis
<figtable id="tab:structure">
Location of mutations in 2nt0_A. Blue: wildtype; Red: mutant; Acc: Solvent accessibility. </figtable>
<figure id="fig:structure_all">
</figure>
Substitution scores
BLOSUM62 scores
The scores were taken from the BLOSUM62 matrix.
<figtable id="tab:subst_blosum">
Nr | Mutation | Score mutation |
Score min |
Score max |
Disease score |
---|---|---|---|---|---|
1 | H99R | 0 | -3 | 8 | 0 |
2 | V211I | 3 | -3 | 4 | -1 |
3 | E150K | 1 | -4 | 5 | 0 |
4 | L236P | -3 | -4 | 4 | 1 |
5 | W248R | -3 | -4 | 11 | 1 |
6 | L509P | -3 | -4 | 4 | 1 |
7 | W351C | -2 | -4 | 11 | 1 |
8 | A423D | -2 | -3 | 4 | 1 |
9 | D482N | 1 | -4 | 6 | 0 |
10 | R83S | -1 | -3 | 5 | 0 |
BLOSUM62 scores of the selected mutations. </figtable>
PSSM of all hits
PSSM.
</figure> </figure><figure id="fig:subst_pssm_all_ali"> |
<figure id="fig:subst_pssm_all"> |
<figtable id="tab:subst_pssm_all">
Position specific substitution scores derived from all significant hits after 5 rounds PSI-BLAST. The respective profile column is shown on the right. </figtable>
PSSM of close homologous sequences
PSSM.
</figure> </figure><figure id="fig:subst_pssm_best_ali"> |
<figure id="fig:subst_pssm_best"> |
<figtable id="tab:subst_pssm_best">
Position specific substitution scores derived from the 60 closest homologous sequences after 5 rounds PSI-BLAST. The respective profile column is shown on the right. </figtable>
Scoring Mutants
SIFT
The predicted results from SIFT Blink is shown here:
Substitution at pos 83 from R to S is predicted to be TOLERATED with a score of 0.17. Median sequence conservation: 2.12 Sequences represented at this position:77 Substitution at pos 99 from H to R is predicted to be TOLERATED with a score of 0.64. Median sequence conservation: 2.14 Sequences represented at this position:80 Substitution at pos 150 from E to K is predicted to be TOLERATED with a score of 0.76. Median sequence conservation: 2.10 Sequences represented at this position:86 Substitution at pos 211 from V to I is predicted to be TOLERATED with a score of 0.56. Median sequence conservation: 2.09 Sequences represented at this position:86 Substitution at pos 236 from L to P is predicted to AFFECT PROTEIN FUNCTION with a score of 0.02. Median sequence conservation: 2.09 Sequences represented at this position:86 Substitution at pos 248 from W to R is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00. Median sequence conservation: 2.09 Sequences represented at this position:86 Substitution at pos 351 from W to C is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00. Median sequence conservation: 2.10 Sequences represented at this position:87 Substitution at pos 423 from A to D is predicted to AFFECT PROTEIN FUNCTION with a score of 0.01. Median sequence conservation: 2.10 Sequences represented at this position:85 Substitution at pos 482 from D to N is predicted to be TOLERATED with a score of 0.69. Median sequence conservation: 2.18 Sequences represented at this position:66 Substitution at pos 509 from L to P is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00. Median sequence conservation: 2.10 Sequences represented at this position:79
The predicted results from SIFT is shown here:
Substitution at pos 83 from R to S is predicted to AFFECT PROTEIN FUNCTION with a score of 0.05. Median sequence conservation: 3.10 Sequences represented at this position:15 Substitution at pos 99 from H to R is predicted to be TOLERATED with a score of 0.74. Median sequence conservation: 3.11 Sequences represented at this position:14 Substitution at pos 150 from E to K is predicted to be TOLERATED with a score of 0.44. Median sequence conservation: 3.10 Sequences represented at this position:16 Substitution at pos 211 from V to I is predicted to be TOLERATED with a score of 1.00. Median sequence conservation: 3.10 Sequences represented at this position:16 Substitution at pos 236 from L to P is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00. Median sequence conservation: 3.10 Sequences represented at this position:16 Substitution at pos 248 from W to R is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00. Median sequence conservation: 3.10 Sequences represented at this position:16 Substitution at pos 351 from W to C is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00. Median sequence conservation: 3.10 Sequences represented at this position:16 Substitution at pos 423 from A to D is predicted to AFFECT PROTEIN FUNCTION with a score of 0.01. Median sequence conservation: 3.10 Sequences represented at this position:16 Substitution at pos 482 from D to N is predicted to be TOLERATED with a score of 0.77. Median sequence conservation: 3.10 Sequences represented at this position:16 Substitution at pos 509 from L to P is predicted to AFFECT PROTEIN FUNCTION with a score of 0.01. Median sequence conservation: 3.11 Sequences represented at this position:14
Polyphen2
H99R This mutation is predicted to be benign with a score of 0.000 (sensitivity: 1.00; specificity: 0.00) This mutation is predicted to be benign with a score of 0.000 (sensitivity: 1.00; specificity: 0.00) V211I This mutation is predicted to be benign with a score of 0.000 (sensitivity: 1.00; specificity: 0.00) This mutation is predicted to be benign with a score of 0.001 (sensitivity: 0.99; specificity: 0.09) E150K This mutation is predicted to be benign with a score of 0.000 (sensitivity: 1.00; specificity: 0.00) This mutation is predicted to be benign with a score of 0.001 (sensitivity: 0.99; specificity: 0.09) L236P This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) W248R This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) This mutation is predicted to be probably damaging with a score of 0.999 (sensitivity: 0.09; specificity: 0.99) L509P This mutation is predicted to be probably damaging with a score of 0.992 (sensitivity: 0.70; specificity: 0.97) This mutation is predicted to be probably damaging with a score of 0.988 (sensitivity: 0.53; specificity: 0.95) W351C This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) A423D This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) This mutation is predicted to be probably damaging with a score of 0.996 (sensitivity: 0.36; specificity: 0.97) D482N This mutation is predicted to be benign with a score of 0.000 (sensitivity: 1.00; specificity: 0.00) his mutation is predicted to be benign with a score of 0.002 (sensitivity: 0.99; specificity: 0.18) R83S This mutation is predicted to be benign with a score of 0.007 (sensitivity: 0.96; specificity: 0.75) This mutation is predicted to be benign with a score of 0.019 (sensitivity: 0.95; specificity: 0.55)
SNAP
Discussion
<figtable id="tab:discussion">
Property | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
---|---|---|---|---|---|---|---|---|---|---|---|
Name | Weight | H99R | V211I | E150K | L236P | W248R | L509P | W351C | A423D | D482N | R83S |
Physicochemical | 1.0 | -1 | -1 | 1 | -1 | 1 | -1 | 1 | 1 | -1 | 0 |
Structure | 0.8 | -1 | -1 | -1 | -1 | 1 | 1 | 0 | 1 | 0 | -1 |
BLOSUM62 | 0.2 | 0 | -1 | 0 | 1 | 1 | 1 | 1 | 1 | 0 | 0 |
PSSM all | 0.4 | 0 | -1 | 0 | 0 | 1 | 1 | 1 | 1 | -1 | 0 |
PSSM close | 0.4 | 0 | -1 | 0 | 1 | 1 | 1 | 1 | 1 | -1 | 0 |
SIFT | 1.0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Polyphen2 | 1.0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
SNAP | 1.0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Average disease score | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | |
Prediction | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | |
Verification | -1 | -1 | 1 | 1 | 1 | -1 | 1 | 1 | -1 | -1 |
Summary of the sequence-based mutation analysis. A final disease score is obtained by computing the weighted average of all individual disease scores. </figtable>
H99R
H99R is not disease causing. Not listed in HGMD.
V211I
V211I is not disease causing. Not listed in HGMD.
E150K
Gaucher disease type 1 [1]
L236P
Gaucher disease type 1 [2]
W248R
Gaucher disease [3]
L509P
L509P is not disease causing. Not listed in HGMD.
W351C
Gaucher disease type 1 [4]
A423D
Gaucher disease [5]
D482N
D482N is not disease causing. Not listed in HGMD.
R83S
R83S is not disease causing. Not listed in HGMD.