Difference between revisions of "Sequence-based mutation analysis"

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!Reliability Index
  
!Reliability Index
 
!Expected Accuracy
  
!Expected Accuracy
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!Cause disease
  
 
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| S/C || 65 || <font color=FF0000>Non-neutral</font> || 3 || 78% || yes
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| S/C || 65 || <font color=FF0000>Non-neutral</font> || 3 || 78%
 
|-
  
|-
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| I/T || 105 || <font color=FF0000>Non-neutral</font> || 3 || 78% || yes
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| I/T || 105 || <font color=FF0000>Non-neutral</font> || 3 || 78%
 
|-
  
|-
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| Q/H || 127 || <font color=FF0000>Non-neutral</font> || 2 || 70% || yes
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| Q/H || 127 || <font color=FF0000>Non-neutral</font> || 2 || 70%
 
|-
  
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| C/Y,S || 282 || <font color=FF0000>Non-neutral</font> || 6/5 || 93%/87% || yes
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| C/Y,S || 282 || <font color=FF0000>Non-neutral</font> || 6/5 || 93%/87%
 
|-
  
|-
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| R/M || 330 || Neutral || 2 || 69% || yes
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| R/M || 330 || Neutral || 2 || 69%
 
|-
  
|-
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| A/V || 176 || <font color=FF0000>Non-neutral</font> || 3 || 78% || yes
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| A/V || 176 || <font color=FF0000>Non-neutral</font> || 3 || 78%
 
|-
  
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| R/S || 6 || <font color=FF0000>Non-neutral</font> || 0 || 58% || yes
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| R/S || 6 || <font color=FF0000>Non-neutral</font> || 0 || 58%
 
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| T/I || 217 || <font color=FF0000>Non-neutral</font> || 1 || 63% || no
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| T/I || 217 || <font color=FF0000>Non-neutral</font> || 1 || 63%
 
|-
  
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| M/T || 35 || Neutral || 1 || 60% || no
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| M/T || 35 || Neutral || 1 || 60%
 
|-
  
|-
 
|}
  
|}

Revision as of 12:52, 27 June 2011

SNP's

Because the HFE-Gen has no annotated functional site, we can just adress the biochemical changes for each SNP. A change in functionality or stability can not be described.
To compare the biochemical properties of the amino acid's, we used the very convenient function of wolfram alpha and the Wikipedia entry about amino acids <ref>http://en.wikipedia.org/wiki/Amino_acid</ref>.

Mutation Position Database Blosum62 PAM1 Pam250 Physicochemical changes
S/C 65 HGMD/dbSNP -1 5 3 There is no change in size and charge, just the polarity changes because an oxygen atom is replaced by a sulfur atom.
I/T 105 HGMD/dbSNP -1 11 6 An OH-group is replaced by an ethyl-group which leads to a small change in size and a large change in hydrophobicity.
Q/H 127 HGMD/dbSNP 0 20 7 Because of the aromatic ring, the flexibility is reduced with a histidine at this position. The polarity and hydrophobicity remain the same.
C/Y,S 282 HGMD/dbSNP -2/-1 3/11 3/7 In both cases, the hydrophobicity is changed. Cysteine is a nonpolar whereas Serine and Tyrosine are polar amino acids.
R/M 330 HGMD -1 1 1 The charge of the side chain changes from positive to neutral and the size is changing but this should not have such a strong impact like the different charge.
A/V 176 HGMD 0 13 9 Alanin and Valine are pretty similar in there properties, just the size changes and the hydrophobicity decreases, but both are water soluble.
R/S 6 HGMD -1 11 6 The polarity is changing which has an impact on the surface on the water solubility of the protein. Also the size of the side chain changes.
T/I 217 dbSNP -1 7 4 An ethyl-group is replaced by an OH-group which leads so a small change in size and a large change in hydrophobicity.
M/T 35 dbSNP -1 6 5 The polarity is changing which has an impact on the surface on the water solubility of the protein.
R/M 58 dbSNP -1 1 1 The charge of the side chain changes from positive to neutral and the size is changing but this should not have such a strong impact like the different charge.
HUMAN_HFE with SNPs from above, HGMD:green, dbSNP:red and both:blue

A change in polarity is just important for residues at the surface of the protein, because with a change in polarity, the hydrophobicity changes and so the water solubility decreases/increases.

Remark:
The reference sequence from UniProt has at position 58 a F, the SNP at this position is annotated as a R to M change. After a comparison of the accession number with the OMIM database, we believe that the position is not correct annotated in dbSNP. The position reported in OMIM is 330, therefore we treated these SNP's as the same and ignored the position 58 SNP. But because we had no SNP's occuring only in dbSNP and HGMD is annotating only disease realated SNP's, we decided to not replace this SNP by another one.

SNP analysis by Hand

First we searched in a non redundant database for homologoues sequences in mammalians. The result list can be found here. We created then a multiple sequence alignment using COBALT to calculate the conservation of the SNP positions.


Mutation Position Conservation(JalView) Secondary Structure (UniProt)
S/C 65 7 Beta-Strand
I/T 105 7 Helix
Q/H 127 2 ---
C/Y,S 282 11 Beta-Strand
R/M 330 6 ---
A/V 176 7 Helix
R/S 6 0 ---
T/I 217 0 ---
M/T 35 0 Beta-Strand

We removed all nonsensical sequences like short ones and sequences with an obvious large insertion or deletion.

Psi-Blast

  • command line: blastpgp -i hfe.fasta -d /data/blast/nr/nr -e 10E-6 -j 5 -Qpsiblast.mat -o psiblast.out
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
   6 R   -2  2 -3 -4 -2 -2 -3 -4 -3  1  4 -2  1  0 -4 -3 -2 -3 -2  1    0  17   0   0   0   0   0   0   0   0  72   0   0   3   0   0   0   0   0   8  0.51 0.24
  35 M    0 -2 -1 -2 -2 -1 -2 -2 -2 -1 -1 -2  3 -2 -2  0  5 -3 -2  0    3   0   1   0   0   0   0   0   0   0   0   0  17   0   0   0  78   0   0   1  0.66 0.25
  65 S    1 -2  0 -1 -2 -1 -1 -1 -2 -3 -3 -1 -2 -3 -2  5  1 -4 -3 -2    1   0   0   0   0   0   0   0   0   0   0   0   0   0   0  99   0   0   0   0  0.80 0.25
 105 I    1 -3 -4 -4 -2 -3 -3 -3 -3  2  4 -3  1 -1 -3 -2 -2 -3 -2  1   19   0   0   0   0   0   0   0   0  17  64   0   0   0   0   0   0   0   0   1  0.50 0.25
 127 Q   -1 -2 -1 -2 -3  2 -1  6 -2 -4 -4 -2 -3 -4 -3 -1 -2 -3 -4 -4    0   0   0   0   0  17   2  81   0   0   0   0   0   0   0   0   0   0   0   0  1.06 0.25
 176 A    5 -2 -2 -2 -1 -1 -1  0 -2 -2 -2 -1 -2 -3 -2  0 -1 -3 -3 -1   96   0   0   0   0   0   4   1   0   0   0   0   0   0   0   0   0   0   0   0  0.72 0.25
 217 T    0 -2  0 -1 -2 -1 -1 -1 -2 -3 -3 -1 -2 -3 -1  5  3 -4 -2 -2    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  83  17   0   0   0  0.70 0.25
 282 C   -1 -4 -4 -4 10 -4 -5 -3 -4 -2 -2 -4 -2 -3 -4 -2 -2 -3 -3 -2    0   0   0   0 100   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  1.89 0.25
 330 R   -3  2 -4 -4  3 -2 -3 -3 -3 -3 -3 -2 -2  0 -4 -3 -3 11  1 -3    0  19   0   0  10   0   0   0   0   0   0   0   0   0   0   0   0  70   1   0  1.90 0.26

SIFT

The prediction of SIFT is marked with a low confidence warning because, the sequences used for the prediction were not diverse enough.

Mutation Position Prediction Score
S/C 65 AFFECT PROTEIN FUNCTION 0.00
I/T 105 AFFECT PROTEIN FUNCTION 0.00
Q/H 127 TOLERATED 0.16
C/Y,S 282 AFFECT PROTEIN FUNCTION 0.00
R/M 330 TOLERATED 0.06
A/V 176 AFFECT PROTEIN FUNCTION 0.01
R/S 6 AFFECT PROTEIN FUNCTION 0.01
T/I 217 TOLERATED 1.00
M/T 35 TOLERATED 1.00

The complete prediction for each position and amino acid can be found here

We got three warning messages form SIFT for which we have no explanation at this time. 

WARNING: Original amino acid H at position 31 is not allowed by the prediction. 
WARNING: Original amino acid S at position 45 is not allowed by the prediction. 
WARNING: Original amino acid Y at position 230 is not allowed by the prediction.

PolyPhen-2

Mutation Position Prediction Score
S/C 65 PROBABLY DAMAGING 0.997
I/T 105 PROBABLY DAMAGING 0.998
Q/H 127 BENIGN 0.002
C/Y,S 282 PROBABLY DAMAGING 1.000/0.997
R/M 330 PROBABLY DAMAGING 0.948
A/V 176 PROBABLY DAMAGING 0.998
R/S 6 PROBABLY DAMAGING 0.738
T/I 217 BENIGN 0.195
M/T 35 PROBABLY DAMAGING 0.989

SNAP

  • command line: snapfun -i hfe.fasta -m muta.txt -o snap.out
Mutation Position Prediction Reliability Index Expected Accuracy
S/C 65 Non-neutral 3 78%
I/T 105 Non-neutral 3 78%
Q/H 127 Non-neutral 2 70%
C/Y,S 282 Non-neutral 6/5 93%/87%
R/M 330 Neutral 2 69%
A/V 176 Non-neutral 3 78%
R/S 6 Non-neutral 0 58%
T/I 217 Non-neutral 1 63%
M/T 35 Neutral 1 60%

Discussion

General

Mutation 1 [R6S]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
R/S 6 Affect Function Affect Function Affect Function --- yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 2 [M35T]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
M/T 35 Tolerated Tolerated Affect Function Beta-Strand no

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 3 [S65C]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
S/C 65 Affect Function Affect Function Affect Function Beta-Strand yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 4 [I105T]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
I/T 105 Affect Function Affect Function Affect Function Helix yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 5 [Q127H]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
Q/H 127 Affect Function Tolerated Tolerated --- yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..


Mutation 6 [A176V]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
A/V 176 Affect Function Affect Function Affect Function Helix yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..


Mutation 7 [T217I]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
T/I 217 Affect Function Tolerated Tolerated --- no

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..


Mutation 8 [C282Y,S]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
C/Y,S 282 Affect Function Affect Function Affect Function Beta-Strand yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..


Mutation 9 [R330M]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure cause disease
R/M 330 Tolerated Tolerated Affect Function --- yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

References

<references />

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