Rs61747114

General Information

SNP-id	rs61747114
Codon	248
Mutation Codon	Leu -> Phe
Mutation Triplet	CTT -> TTT

Back to [Sequence-based mutation analysis]

Sequence-based Mutation Analysis

Pysicochemical Properties

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, whereas 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 does not change the structure of the protein that much. Therefore, we suggest it is possible, that the protein works.

Back to [Sequence-based mutation analysis]

---

Visualization of the Mutation

In the next step, we created the visualization of the mutation with PyMol. Therefore we created a picture for the original amino acid (Figure 1), for the new mutated amino acid (Figure 2) and finally for both together in one picture whereas the mutation is white colored (Figure 3). 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 aromatic ring. The only thing that agrees is the orientation and the length of the residues before the fork or the aromatic ring. All in all the difference probably prevails the agreement and therefore the mutation will probably cause changes in protein structure and function.

picture original amino acid	picture mutated amino acid	combined picture
Figure 1: Amino acid Leucine	Figure 2: Amino acid Phenylalanine	Figure 3: Picture which visualize the mutation

Back to [Sequence-based mutation analysis]

---

Substitution 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 this 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 from 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 both PAMs are not really significant and therefore we are not able to determine effects on the protein for these 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 amino acid	most frequent substitution	rarest substitution	value amino acid	most frequent substitution	rarest substitution	value amino acid	most frequent substitution	rarest substitution
13	45 (Met)	0 (Asp, Cys)	13	20 (Met)	2 (Cys)	0	2 (Ile, Met)	-4 (Asp, Gly)

Back to [Sequence-based mutation analysis]

---

PSSM Analysis

Besides, we looked additional at the position specific scoring matrix (PSSM) for our 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 position is likely very uncommon which indicates that this substitution has bad effects as a consequence. Therefore, we concluded that this mutation will probably cause protein structure changes as well as functional changes.

PSSM
value amino acid	most frequent substitution	rarest substitution
-3	3	-5

Back to [Sequence-based mutation analysis]

---

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 at this position, which can be seen in Figure 4. The regarded mutation is presented by the second colored column. Here we can see, that all the other mammalians have also the amino acid Leucine on this position. Therefore, the amino acid at this position is highly conserved and a mutation there will cause probably huge structural and functional changes for the protein.

Figure 4: Mutation in the multiple alignment

Back to [Sequence-based mutation analysis]

---

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 drastic the mutation can be. In this case both tools agree and predict at the position of the mutation a coil. This has as 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 drastic change of the protein structure and its function is unlikely because the mutation does not affect a secondary structure 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 mutation (red) in the real 3D-structure of PDB and compare it with the predicted secondary structure (Figure 5 and Figure 6). The visualization 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 mutation position disagrees with the position of the predicted secondary structure and is within an alpha-helix. This means a mutation will probably destroy or split the alpha helix which affects drastic 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 extreme changes.

Figure 5: Mutation at position 248

Figure 6: Mutation at position 248 - detailed view

Back to [Sequence-based mutation analysis]

---

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 as a result that the exchange 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]

Back to [Sequence-based mutation analysis]

---

SIFT Prediction

Next, we used SIFT Prediction which displays if a mutation is neutral or not. Therefore, it first shows a row which contains a score for the particular mutation position to a certain amino acid. The amino acid which are not tolerated at this position are colored red. Besides, it also constructs a table which lists the amino acids that are predicted as tolerated and not-tolerated (compare Figure 8).

In this case, there are seven substitutions that are tolerated: Tyrosine, Isoleucine, Aspartic acid, Valine, Phenylalanine, Methionine and Leucine. The substitution to Phenylalanine is tolerated at this position. This means that this mutation at this position is probably neutral and will not cause any structural and function changes of the protein.

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.

Figure 7: Legend

Figure 8: SIFT Table Threshold for intolerance is 0.05. Amino acid color code: non-polar, uncharged polar, basic, acidic. Capital letters indicate amino acids appearing in the alignment, lower case letters result from prediction.

Predict Not Tolerated Position Seq Rep Predict Tolerated c w p e n k g r s t h a Q 248L 1.00 Y I D V F M L

Back to [Sequence-based mutation analysis]

---

PolyPhen2 Prediction

Finally, we also regarded the PolyPhen2 prediction for this mutation. This prediction visualizes how strongly damaging the mutation probably will be. Therefore it gives the result for two possible cases: HumDiv and HumVar. HumDiv is the preferred model for evaluation rare allelse, dense mapping of regions identified by genome-wide association studies and analysis of neutral selection. In contrast, HumVar is the preferred model for diagnostic of Mendelian diseases which require distinguishing mutations with drastic effects from all remaining human variations including abundant mildly deleterious alleles. We decided to look at both possible models, which agreed the most cases.

In this case, the models disagree. The first model HumDic predicts that the mutation is benign (which could be seen on figure 9) whereas the second model HumVar predicts that the mutation is possibly damaging (compare Figure 10). 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 these two results that the mutation is probably neutral and will cause no damage.

Figure 9: HumDiv prediction

Figure 10: HumVar prediction

Back to [Sequence-based mutation analysis]

Structure-based Mutation Analysis

Mapping onto Crystal Structure

Figure 11: Visualization of the mutation and important functional sites
Color declaration:
* red: position of mutation
* green: position of active side
* yellow: position of glycolysation
* cyan: position of Cysteine

First of all, we colored the important residues and also the mutated residue in the crystal structure, to see if the mutation is near of far away from the functional residues. As you can see on Figure 11, the mutation is located within a helix but far away from the functional residues. The mutation is located at the end or begin of a helix. Therefore, of course it is possible that a mutation of this residue causes changes in the structure of the protein. But it is at the side of a helix and therefore the damage should not be that dramatically.

Back to [Structure-based mutation analysis]

---

SCWRL Prediction

Next, we analysed the mutation in more detail. Therefore we looked directly to the structure and orientation of the residue.

picture original amino acid	picture mutated amino acid	combined picture
Figure 12: Amino acid Leucine	Figure 13: Amino acid Phenylalanine	Figure 14: Picture which visualize the mutation

On the pictures we can see, that the amino acids themselves are very different (Figure 12 and Figure 13), but also the location and orientation of the amino acids differ dramatically. Furthermore the two amino acids have a totally different structure (Figure 14) and it is very likely that the properties of these two amino acids differ extremely. Therefore it is very likely, that this mutation destroys the helix structure.

Back to [Structure-based mutation analysis]

---

FoldX Energy Comparison

One important point in the analysis of mutations is to look at the energy of the protein with the original and with the mutated amino acid. Often the energy increases dramatically with the mutated amino acid. This means, that the protein becomes very unstable and therefore, it is often possible that the protein can not bind its ligands any longer. Otherwise, it is also possible, that the protein with the mutated amino acid has a lower energy than the original protein. This means, that the protein is too rigid and loses its flexibility. Than it is also possible, that the protein can not bind the ligands any longer.

Therefore, we compared the energy of our protein with different methods. Here we want to present the result of FoldX.

Original total energy	Total energy for the mutated protein	Strongest energy changes within the mutated protein
-154.17	-153.78	-

The energy of the mutated structure is a little bit higher than the energy of the original structure, but the difference is not that much. To have the possibility to compare the energy values with values from other programs, we calculated a ratio between these two values.

Ratio of the original structure	Ratio of the mutated structure	Difference
100	99.85	0.15

Back to [Structure-based mutation analysis]

---

Minimise Energy Comparison

Next we use the minimise energy tool to compare the energy values of the two different structures.

Comparing Energy:

Original total energy	Total energy for the mutated protein
-9610.467157	-9606.588566

As before, in this case the mutated structure has a higher energy than the original structure. But again, the difference between the two energy values is not very high. To have the possibility to compare the energy values of the different tools, we calculate again the ratio between these two values.

Ratio of the original structure	Ratio of the mutated structure	Difference
100	99.96	0.04

As before, the ratio between these two energy values is very low.

Comparing Structure:

This tool also gives as output a PDB file with the position of the original and the mutated amino acid.

picture original amino acid	picture mutated amino acid	combined picture
Figure 15: Amino acid Leucine	Figure 16: Amino acid Phenylalanine	Figure 17: Picture which visualize the mutation

Again we can see that these two amino acids (Figure 15, Figure 16 and Figure 17) differ extremely in location and orientation within the protein. The result is the same as we got wit SCWRL (Figure 12, Figure 13 and Figure 14).

Visualization of H-bonds and Clashes:

To get more insight in the effects of the mutated amino acid on the structure, we also analysed the H-bonds and clashes of the Asparagine residue.

H-bonds of the original structure	H-bonds near the mutation	Clashes of the mutation
Figure 18: H-bonds of the original amino acid (colored in magenta)	Figure 19: H-bonds of the mutated amino acids (colored in red)	Figure 20: Possible clashes

If we compare the H-Bonds of the original (Figure 18) and the mutated amino acids (Figure 19), we can see that there are no H-Bonds between the residues and the rest of the protein. Therefore the damage of the protein is not caused by missing H-Bonds. Furthermore, we can see that there are no clashes in the protein if the amino acid was mutated (Figure 20).

Back to [Structure-based mutation analysis]

---

Gromacs Energy Comparison

The last tool we used for the energy analysis was Gromacs.

Comparing Energy:

To analyse the energy values calculated by Gromacs, we used the AMBER99SB-ILDN force field.

Here are the values of the original structure:

Energy	Average	Err.Est.	RMSD	Tot-Drift
Bond	1091.57	270	-nan	-1622.75
Angle	3326.81	62	-nan	404.076
Potential	-61304.1	960	-nan	-6402.44

Here you can see the values which gromacs calculated for the structure with the mutated amino acid:

Energy	Average	Err.Est.	RMSD	Tot-Drift
Bond	920.202	180	2165.56	-1059.94
Angle	3202.18	61	233.689	406.391
Potential	-48802.5	830	3227.37	-5533.99

One difference between gromacs and the other tools we used is, that gromacs also calculated the energy for the bonds and the angles. To compare the energies between the different tools we only consider the potential energy in our analysis, because the potential energy is the energy of the complete protein. Therefore, we calculated the ratio between the energies only for the potential energy.

Ratio of the original structure	Ratio of the mutated structure	Difference
100	79.60	20.40

In comparison with the other two methods we used for the energy analysis the difference between the gromacs energy values is much higher.

Comparing Structure:

picture original amino acid	picture mutated amino acid	combined picture
Figure 21: Amino acid Leucine	Figure 22: Amino acid Phenylalanine	Figure 23: Picture which visualize the mutation

As we saw before, the two amino acids differ extremely.

Visualization of H-bonds and Clashes:

Gromacs also offers the user some pictures with the mutated amino acid.

H-bonds of the original structure	H-bonds near the mutation	Clashes of the mutation
Figure 24: H-bonds of the original amino acid (colored in magenta)	Figure 25: H-bonds of the mutated amino acid (colored in red)	Figure 26: Possible clashes

If we look at the pictures (Figure 24, Figure 25) we can see, that there are no H-bonds between the amino acids (the original and mutated) and the protein. Therefore, a damage of the protein can not be explained by missing or additional H-Bonds. Furthermore, it is not possible to find any clashes between the mutated amino acid and the rest of the protein, what can be seen in Figure 26.

Back to [Structure-based mutation analysis]

---