Gaucher Disease: Task 08 - LabJournal

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Choosing Mutation set

The cDNA sequence used by HGMD has the accession number NM_001005741.2. The one letter code of this cDNA has a 100% sequence identity to the reference sequence of UniProt, which we used for all tasks. Therefore, the exact positions of the mutations listed by HGMD can be taken. This accession number is also listed by dbSNP, so that the right mutation position can be seen.

To choose mutations from dbSNP we selected four point mutations of the SNP Geneview Report of glucocerebrosidase with the NP_001005741.1 (corresponding protein isoform of the transcript NM_001005741.2) which was already used in task 7. For HGMD we randomly picked six mutations from the SNP list of the NP_001005741.1.

Mutation Analysis

The property information about the amino acids were taken from Wikipedia.

The visualisation of the mutations was done with pymol. For the mutation of the residues we followed the description of the PymolWiki We considered again the sequence shift of 39 residues between uniprot and pdb sequence.

We took the information about the secondary structure from our previous task 3. We alway choosed the secondary structure type which was predicted from the majority of the prediction tools in that task.

The scores for the mutations were looked up in two substitution matrices: BLOSUM62 and PAM250

PSSM

We created different PSSM matrices. The first was generated with PsiBlast:

blastpgp -j 5 -h 10e-6
-i /mnt/home/student/gerkej/gaucher/task8/P04062.fasta 
-d /mnt/project/pracstrucfunc13/data/big/big_80  
-o /mnt/home/student/gerkej/gaucher/task8/psiBl/ev-6/big80_it5.out 
-Q /mnt/home/student/gerkej/gaucher/task8/psiBl/ev-6/big80_it5.pssm 
-C /mnt/home/student/gerkej/gaucher/task8/psiBl/ev-6/big80_it5.chk

PSSM by psiblast

Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
          A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V    A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
  77 S    0  1  0  1 -1  1  1 -1  0 -1 -2  0 -1 -3 -3  1  1 -4 -2  0    7   9   4   8   1   7  11   3   2   4   4   6   1   1   0  11  11   0   1   8  0.14 inf
 141 N   -1  0  2  4 -2  2  2 -3 -1 -3 -3  1 -2 -5 -4  0  1  1 -2 -3    5   4  10  20   1  10  15   2   1   1   3   7   1   0   0   7   7   2   1   2  0.40 inf
 159 R   -6  8 -6 -7 -8 -4 -5 -7 -6 -8 -4  2 -6 -7 -7 -3 -6  1 -7 -7    0  81   0   0   0   0   0   0   0   0   2  11   0   0   0   2   0   2   0   0  2.56 inf
 213 L   -3 -5 -2 -6 -4 -5 -5 -2 -5  4  2 -5  0  3 -5 -4 -1  3  0  3    2   0   2   0   0   0   0   3   0  23  22   0   2  13   0   0   4   4   2  21  0.72 inf
 241 G   -1  2  0  2 -4  1 -1  1 -1 -2 -1  3 -2 -3 -1 -1 -1  0 -1 -2    5  11   5  11   0   7   3  11   2   2   6  17   1   1   4   3   4   2   3   3  0.19 inf
 349 V    0 -3 -2 -3  0 -4 -3  2 -1  0 -1 -4 -1  4 -5  0  0  5  2  2   10   1   1   1   2   0   1  14   2   5   4   0   2  17   0   6   6   9   7  14  0.42 inf
 408 T   -1  1  2  2  0  2  0  2  2 -2 -2  0 -1 -1 -1  0  0 -4  0 -2    3   7  10  11   2   9   5  19   5   2   3   4   2   2   2   5   4   0   3   3  0.19 inf
 409 N    1 -2  1  1  1 -2 -1  0  1 -1 -1 -1  0  1  0  1  0  2  1 -1   11   0  10   9   4   0   3   6   3   4   4   3   3   6   4   9   6   4   7   4  0.09 inf
 483 L   -2 -3 -2 -3 -4 -3 -3  0 -2  3  3 -2  0  0 -3 -3 -2 -4 -1  3    2   1   2   2   0   1   1   9   1  13  29   3   2   4   1   2   2   0   2  24  0.46 inf
 501 N   -6 -6  9 -3 -8 -4 -3 -6 -5 -6 -8 -6 -3 -8 -5 -2 -2 -8 -7 -6    1   0  87   1   0   0   2   0   0   0   0   0   1   0   1   3   3   0   0   1  2.83 inf

The second PSSM is based on an alignment of all mamalian homologous. We identified the homologs on Uniprot. The BlastP seaerch was run on the mamalian database. For P04062 we found 140 homologous sequences with an evalue of less than 10e-4. To generate the MSA of the homologous sequences we used Clustal Omega, which is a newer version of CLustalW and recommended on the ClustalW webserver. With PsiBlast we created the PSSM matrix out of the MSA.

blastpgp 
-i /mnt/home/student/gerkej/gaucher/task8/P04062.fasta 
-d /mnt/project/pracstrucfunc13/data/big/big_80  
-B /mnt/home/student/gerkej/gaucher/task8/psiBl/clustal-omegas.clustal
-o /mnt/home/student/gerkej/gaucher/task8/psiBl/big80_it5.out 
-Q /mnt/home/student/gerkej/gaucher/task8/psiBl/big80_it5.pssm 

PSSM by clustalOmega alignment of hoologous

Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
          A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V    A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
  77 S    1 -1  0  0 -1  0  0  0 -1 -2 -2  0 -1 -2  0  3  1 -2 -1 -1    3   2   2   2   1   1   2   4   1   2   3   2   1   1   2  64   4   0   1   2  0.25 inf
 141 N   -1  0  5  3 -2  0  1 -1  0 -3 -3  0 -2 -3 -1  0  0 -3 -2 -2    1   1  55  16   0   1  10   1   0   1   1   6   0   1   1   3   1   0   0   1  0.42 inf
 159 R   -2  5 -1 -2 -3  1  0 -3 -1 -3 -2  2 -2 -2 -2 -1 -1  5 -1 -3    0  86   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  14   0   0  0.76 inf
 213 L   -2 -2 -4 -4 -1 -2 -3 -4 -3  2  4 -3  2  0 -3 -3 -1 -2 -1  1    0   0   0   0   0   0   0   0   0   0 100   0   0   0   0   0   0   0   0   0  0.49 inf
 241 G    0  1 -1 -1 -3 -1 -2  5 -2 -4 -4 -1 -3 -3 -2  0 -2 -3 -3 -3    0  17   0   0   0   0   0  83   0   0   0   0   0   0   0   0   0   0   0   0  0.83 inf
 349 V    0 -3 -3 -3 -1 -2 -3 -3 -3  3  1 -2  1 -1 -3 -2  0 -3 -1  4    0   0   0   0   0   0   0   0   0   3   0   0   0   0   0   0   0   0   0  97  0.39 inf
 408 T    0 -1  0 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -2 -1  1  4 -2 -1  0    1   1   1   1   0   1   1   1   0   1   2   1   0   1   1   1  82   0   1   1  0.37 inf
 409 N   -1  0  5  1 -2  0  0  0  0 -2 -2  0 -2 -2 -1  0  0 -3 -2 -2    2   1  76   5   0   1   1   2   0   1   2   1   0   1   1   2   1   0   1   1  0.51 inf
 483 L   -2 -2 -4 -4 -1 -2 -3 -4 -3  2  4 -3  2  0 -3 -3 -1 -2 -1  1    0   0   0   0   0   0   0   0   0   0 100   0   0   0   0   0   0   0   0   0  0.50 inf
 501 N   -1  0  6  1 -2  0  0  0  0 -3 -3  0 -2 -3 -2  0  0 -3 -2 -2    1   1  86   1   0   1   1   1   0   1   1   1   0   1   1   1   1   0   0   1  0.61 inf

Prediction approaches

SIFT

SIFT was run on the webserver. We got the following predictions for our mutations. The score ranges between 0 and 1. A score <=0.05 indicated to an disease causing amino acid substitution. Otherwise, the substitution is "tolerated" which means that the mutation has no effect on the protein function.

S77R

Substitution at pos 77 from S to R is predicted to be TOLERATED with a score of 0.37.
   Median sequence conservation: 3.10
   Sequences represented at this position:15

N141S

Substitution at pos 141 from N to S is predicted to be TOLERATED with a score of 0.15.
   Median sequence conservation: 3.13
   Sequences represented at this position:15

R159Q

Substitution at pos 159 from R to Q is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.10
   Sequences represented at this position:16

L213F

Substitution at pos 213 from L to F is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.10
   Sequences represented at this position:16

G241E

Substitution at pos 241 from G to E is predicted to AFFECT PROTEIN FUNCTION with a score of 0.01.
   Median sequence conservation: 3.10
   Sequences represented at this position:16

V349I

Substitution at pos 349 from V to I is predicted to be TOLERATED with a score of 0.25.
   Median sequence conservation: 3.10
   Sequences represented at this position:16

T408M

Substitution at pos 408 from T to M is predicted to AFFECT PROTEIN FUNCTION with a score of 0.03.
   Median sequence conservation: 3.10
   Sequences represented at this position:16

N409

Substitution at pos 409 from N to S is predicted to be TOLERATED with a score of 0.05.
   Median sequence conservation: 3.10
   Sequences represented at this position:16

L483P

Substitution at pos 483 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

N501S

Substitution at pos 501 from N to S is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.10
   Sequences represented at this position:16

Polyphen2

Polyphen2 offers two different prediction scores which were trained and tested on different datasets. As recommended on the website, we decided to focus on the score HumVar which uses all human disease-causing mutations from UniProtKB and common human non-synonymous SNPs as non-damaging SNPs. For the Polyphen2 nomenklatur benign, possibly damaging and probably damaging we used the identifier non-disease causing, possibly damaging and disease causing.

The score ranges between 0 and 1 and reflects the probability for the SNP having a damaging effect.

S77R
This mutation is predicted to be benign with a score of 0.170 (sensitivity: 0.89; specificity: 0.72)
N141S
This mutation is predicted to be benign with a score of 0.009 (sensitivity: 0.96; specificity: 0.49)
R159Q
This mutation is predicted to be probably damaging with a score of 0.997 (sensitivity: 0.27; specificity: 0.98)
L213F
This mutation is predicted to be possibly damaging with a score of 0.790 (sensitivity: 0.76; specificity: 0.87)
G241E
This mutation is predicted to be possibly damaging with a score of 0.892 (sensitivity: 0.70; specificity: 0.90)
V349I
This mutation is predicted to be benign with a score of 0.118 (sensitivity: 0.90; specificity: 0.70)
T408M
This mutation is predicted to be benign with a score of 0.113 (sensitivity: 0.90; specificity: 0.69)
N409S
This mutation is predicted to be benign with a score of 0.234 (sensitivity: 0.88; specificity: 0.75)
L483P
This mutation is predicted to be possibly damaging with a score of 0.856 (sensitivity: 0.72; specificity: 0.88)
N501S
This mutation is predicted to be probably damaging with a score of 0.979 (sensitivity: 0.57; specificity: 0.94)

MutationTaster

We used the MutationTaster web version. As input we used:

  • Gene: GBA
  • Transcript: ENST00000327247 (protein_coding, 2387 bases) NM_001005741
  • Position/snippet refers to coding sequence (ORF)

For the alteration, we calculated the position of the mutated bases.

The score represents the probability for the prediction.

S77R

Prediction: disease causing
Model: simple_aae, prob: 0.897766357553113 
Summary: amino acid sequence changed, protein features (might be) affected, splice site changes

N141S

Prediction: polymorphism
Model: simple_aae, prob: 0.828715180775458      
Summary: amino acid sequence changed, protein features (might be) affected, splice site changes

R159Q

Prediction: disease causing
Model: simple_aae, prob: 0.999998729728442      
Summary: amino acid sequence changed, known disease mutation at this position (HGMD CM880035), known disease mutation: rs79653797 (pathogenic), protein features (might be) affected, splice site changes

L213F

Prediction: disease causing
Model: simple_aae, prob: 0.99997844587523      
Summary: amino acid sequence changed, known disease mutation at this position (HGMD CM057076), protein features (might be) affected, splice site changes

G241E

Prediction: disease causing
Model: simple_aae, prob: 0.999988349653367      
Summary: amino acid sequence changed, known disease mutation at this position (HGMD CM992894), protein features (might be) affected, splice site changes

V349I

Prediction: disease causing
Model: simple_aae, prob: 0.998220211902893      
Summary: amino acid sequence changed, protein features (might be) affected, splice site changes

T408M

Prediction: polymorphism
Model: simple_aae, prob: 0.595642579948168      
Summary: amino acid sequence changed, heterozygous in TGP, known disease mutation at this position (HGMD CM960697), protein features (might be) affected, splice site changes

N409S

Prediction: disease causing
Model: simple_aae, prob: 0.998851047063661       
Summary: amino acid sequence changed, known disease mutation at this position (HGMD CM880036), protein features (might be) affected, splice site changes

L483P

Prediction: disease causing
Model: simple_aae, prob: 0.999999999939023 
Summary: amino acid sequence changed, known disease mutation at this position (HGMD CM870010, HGMD CM940819, rs421016 (pathogenic)), protein features (might be) affected, splice site changes


N501S

Prediction: disease causing
Model: simple_aae, prob: 0.999999999990917     
Summary: amino acid sequence changed, known disease mutation at this position (HGMD CM057072), protein features (might be) affected, splice site changes

SNAP2

For SNAP we created a File /.snap2rc with the pathes of all databases needed by SNAP. After that SNAP2 was run on the rostlab server as followed

snapfun -i P04062.fasta -m mutation-list.txt -o snap_P04062.out

nsSNP        Prediction        Reliability Index        Expected Accuracy
-----        ------------        -------------------        -------------------
S77R          Neutral                 2                        69%
N141S         Neutral                 5                        89%
R159Q         Non-neutral             7                        96%
L213F         Neutral                 0                        53%
G241E         Neutral                 0                        53%
V349I         Neutral                 5                        89%
T408M         Neutral                 5                        89%
N409S         Neutral                 0                        53%
L483P         Neutral                 1                        60%
N501S         Non-neutral             0                        58%