Sequence-based mutation analysis

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Revision as of 14:11, 27 June 2011 by Greil (talk | contribs) (→‎Mutation 8 [C282Y,S])

SNP's

HUMAN_HFE with SNPs from above, HGMD:green, dbSNP:red and both:blue

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>. All SNPs are visualized at the following picture of the HFE_HUMAN UniProt sequence.

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.

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

SIFT (Sorting Intolerant From Tolerant) uses the knowledge, that important amino acids are more conservered and therefore less mutable than non important amino acids. It predicts if the mutation affects the protein function or not. SIFT was created in 2003 by Ng P. and Henikoff S.

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

PolyPhen-2 (Polymorphism Phenotyping Version 2) uses structure and sequence based features to predict the influence of an single nucleotide mutation at an human protein. PolyPhen-2 was established by Adzhubei et al. in 2010.

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

SNAP (screening for non acceptable polymorphisms) uses an neural network approach to predict the functional effects of non-synonymous SNPs. It was developed by Rost B. and Bromberg Y. in 2007. It is installed locally at our VM and therefore useable with the command line.

  • 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

We tried to gain as much knowledge of the SNP and therefore combined the information of the mutated amino acids, results from SNAP, SIFT and PolyPhen2 and if the mutation occurs inside an secondary structure element. We decided to to introduce the field HGMD/dbSNP disease with the groundtruth according to HGMD or dbSNP, if that mutation causes the disease. We are going to discuss each SNP one by one and will finally give an conclusion based on our opinion whether the mutation is disease causing or not. This statement will be compared against the groundtruth from HGMD/dbSNP.

Mutation 1 [R6S]

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

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.

Mutation 2 [M35T]

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

It is the mutation from the nonpolar and neutral Cysteine to the polar and neutral Tyrosine or Serine, therefore a change in the hydrophobocity occurs. Thyrosine also forms a ring and can influence the protein folding. The change occurs inside an beta-strand which is also bad, because changes inside of secondary structure elements might affect the structure of the protein and thus the function.

The substitution matrices values are high for the change of Cysteine to Serine and low for the change to Thyrosine. That indicates, the replacement with Serine is rare and the change to Thyrosine occurs often. According to JalView is the conversation very high.

SNAP, SIFT and PolyPhen2 came to the conclusion, that the change affects the function of the protein. Looking also at the other results, we came to the same conclusion, because all indicates and breakdown of the protein function.

According to HGMD is our assumption correct, the C282Y,S mutation is the most commonly occuring mutation and causes around 90% of all hemochromatosis cases.

Mutation 9 [R330M]

Mutation Position SNAP SIFT PolyPhen2 Secondary Structure HGMD/dbSNP 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

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