Rs61747114
Contents
General Information
| SNP-id | rs61747114 |
| Codon | 248 |
| Mutation Codon | Leu -> Phe |
| Mutation Triplet | CTT -> TTT |
Pysicochemical Properities
First of all, we explored the amino acid properties and compared them for the original and the mutated amino acid. Therefore we created the possible effect that the mutation could have on the protein.
| Leu | Phe | consequences |
| aliphatic, hydrophobic, neutral | aromatic, hydrophobic, neutral | Leu is an aliphatic amino acid, wheras Phe is an aromatic amino acid. This means, that Phe has an aromatic ring in its structure. But both amino acids are relatively big and so it is possible, that the exchange of this amino acids do not change the structure of the protein that much. Therefore, we suggest it is possible, that the protein will work. |
Visualisation of the Mutation
In the next step, we created the visualization of the muation with PyMol. Therefore we created a picture for the original amino acid, for the new mutated amino acid and finally for both together in one picture whereas the mutation is white colored. The following pictures display that the mutated amino acid Phenylalanine looks very different to Leucine. Leucine forks at the end of the rest whereas Phenylalanine has an huge aromatical ring. The only thing that agrees is the orientation and the length of the res before the fork or the aromativ ring. All in all the difference probably prevails the agreement and therefore the mutation will probably caus changes in protein structure and function.
| picture original aa | picture mutated aa | combined picture |
Subsitution Matrices Values
Afterwards, we looked at the values of the substitution matrices PAM1, PAM250 and BLOSSUM62. Therefore we looked detailed at the three values: the value for accoding amino acid substitution, the most frequent value for the substitution of the examined amino acid and the rarest substitution.
In this case, the substitution of Leucine to Phenylalanine has average values that are almost as far away from the rarest value as to the most frequent value for all three substitution matrices. Only BLOSOUM62 is a little bit closer to the most frequent substitution. Therefore, the values from from both PAMs are not realy significant and therefore we are not able to determine effects on the protein for this two matrices. Otherwise, according to BLOSOUM62 a mutation at this position will probably not cause structural changes which can affect functional changes.
| PAM 1 | Pam 250 | BLOSOUM 62 | ||||||
| value aa | most frequent substitution | rarest substitution | value aa | most frequent substitution | rarest substitution | value aa | most frequent substitution | rarest substitution |
| 13 | 45 (Met) | 0 (Asp, Cys) | 13 | 20 (Met) | 2 (Cys) | 0 | 2 (Ile, Met) | -4 (Asp, Gly) |
PSSM Analysis
Besides, we looked additional at the position specific scoring matrix (PSSM) for ouer sequence. In contrast to PAM and BLOSOUM, the PSSM contains a specific substitution rate for each position in the sequence. Therefore, the PSSM is more position specific than PAM or BLOSOUM. We extracted the substitution value for the underlying mutation, the value for the most frequent substitution and the rarest substitution.
In this case the substitution rate for Leucine to Phenylalanine at this position is very low and near the value for the rarest substitution. This means this substitution at this positin is likely very uncommon which indicates that this substitution has bad effects as a consequence. Therefore, we conclude that this mutation will probably cause protein structure changes as well as functional changes.
| PSSM | ||
| value aa | most frequent substitution | rarest substitution |
| -3 | 3 | -5 |
| self-information | expected self-information | |
| Leu | 1 | 13 |
| Phe | -3 | 1 |
Conservation Analysis with Multiple Alignments
As a next step we created a multiple alignment which contains the HEXA sequence and 9 other mammalian homologous sequences from uniprot. Afterwards we looked at the position of the different mutations and looked at the conservation level on this position. The regarded mutation is presented by the second colored column. Here we can see, that the all other mammalians have also on this position the amino acid Leucine. Therefore, the mutation on this position is highly conserved and a mutation there will cause probably huge structural and functional changes for the protein.
Secondary Structure Mutation Analysis
As a next step we compared the different results of the secondary structure prediction tools JPred and PsiPred. Afterwards we can examine in which secondary structure element and where therein the mutation takes place. This can give an overview of how drastical the mutation can be. In this case both tools agree and predict at the position of the mutation a coil. This has a result, that the mutation at this position would not destroy or split a secondary structure element. It will probably only changes the coil between two secondary structure elements, but this can sometimes also cause a change of the the following secondary structure. We think that a drastical change of the protein structure and its function is unlikly because the mutation does not affect a secondary struture element. The change of the coil will probably only take places between two secondary structure elements which will probably not change.
JPred: ...HHHHHHHHCCCEEEECCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCC... PsiPred: ...HHHHHHHHCCCEEEECCCCCHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCH...
Comparison with the real Structure:
Afterwards we also visualize the position of the muation (red) in the real 3D-structure of PDB and compare it with the predicted secondary structure. The visualisation can therefore like above the predicted secondary structure display if the mutation is in a secondary structure element or in some other regions.
Here in this case the mutationposition disagree with the position of the predicted secondary structure and is within a alpha-helices. This means a mutation will probably destroy or split the alpha helix which affects drastical structural changes on the protein. We think that a structural change is very likely, because it is within a secondary structure element and will therefore cause extrem changes.
SNAP Prediction
Next, we looked at the result of the SNAP prediction. For this prediction we took the amino acid of the certain position and checked every possible amino acid mutation. Afterwards we extract the result for Phenylalanine which is the real mutation in this case. SNAP has a result that the exhange from Leucine to Phenylalanine at this position is neutral with a relative high accuracy. This means that this certain mutation on this position cause very likely no structural and functional changes of the protein.
| Substitution | Prediction | Reliability Index | Expected Accuracy |
| F | Neutral | 3 | 78% |
A detailed list of all possible substitutions can be found [here]
SIFT Prediction
SIFT Matrix:
Each entry contains the score at a particular position (row) for an amino acid substitution (column). Substitutions predicted to be intolerant are highlighted in red.
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SIFT Table
Threshold for intolerance is 0.05.
Amino acid color code: nonpolar, uncharged polar, basic, acidic.
Capital letters indicate amino acids appearing in the alignment, lower case letters result from prediction.
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PolyPhen2 Prediction
Finally, we also regarded the PolyPhen2 prediction for this muation. This prediction visualizes have strongly demaging the mutation probably will be. Therefore it gives the result for two possible cases: HumDiv and HumVar. HumDiv is a prefered model for evaluation rare allels, dense mapping of regions identified by genome-wide assiociation studies and analysis of neutral selection. In contrast, HumVar is a prefered model for diagnostic of Mendelian diseases which require distinguishing mutations with drastic effects from all remaining human variations including abundant mildly deleterious allels. We decided to look at both possible models, which are agrees in the most cases.
In this case, the models disagree. The first model HumDic predicts that the mutation is benign whereas the second model HumVar predicts that the mutation is possibly damaging. When we look at the scores we can see that the difference is with 0.213 not so high, which displays that they are moving in the same direction. Looking at both together, there is probably no huge structural change but with a small probability there can possibly be a damage of the protein. We assume with the help of this two results that the mutation is probably neutral and will cause no damage.
