Rs121907968

General Information

SNP-id	rs121907968
Codon	485
Mutation Codon	Trp -> Arg
Mutation Triplet	gTGG -> CGG

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Sequence-based Mutation Analysis

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.

Trp	Arg	consequences
aromatic, polar, hydrophobic, neutral	positive charged, polar, hydrophilic	Trp is very big, because of two aromatic rings in its structure. Furthermore, it is hydrophobic, whereas, Arg is a hydrophilic amino acid. Therefore, the changes in the 3D structure might be extreme and delete the function of the protein.

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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 Arginine looks very different to Tryptophan. Tryptophan has two huge aromatical rings. Contrary, Aspartate is also a long amino acid and forks at the end of the rest. Furthermore, Arginige is also orientated in a completly different direction. Because of the aromatical rings the differences between these two amino acids is realy huge. Therefore, the amino acids will probably cause drastical effects on protein structure and function.

picture original aa	picture mutated aa	combined picture
Amino acid Tryptophan	Amino acid Arginine	Picture which visualize the mutation

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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 Tryptophan to Arginine has very high values that agree with the most frequent value for PAM1 and PAM250. In contrast, BLOSOUM62 has a low value for this substitution which is near the value for the rarest substitution. The difference between the two PAMs and BLOSUM62 can be ascribed to the different preparations of these two kind of substitutions matrices. The PAM-matrices is an evolutionary model whereas BLOSUM is based on protein families. Therefore probably this mutation is evolutionary not that unlikely whereas within a protein family it is very unusual. Therefore, according to PAM1 and PAM250 a mutation at this position will probably not cause structural changes which can affect functional changes whereas according to BLOSSUM62 a mutation at this position will probably cause structural and functional changes of the protein.

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
2	2 (Arg)	0 (all, except Arg, Phe, Ser, Tyr)	2	2 (Arg)	0 (all, except Arg, His, Leu, Phe, Ser, Tyr)	-3	2 (Tyr)	-4 (Asn, Asp, Pro)

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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 Tryptophan to Arginine acid 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 conclude that this mutation will probably cause protein structure changes as well as functional changes.

PSSM
value aa	most frequent substitution	rarest substitution
-6	13	-7

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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 colored column. Here we can see, that the all other mammalians have also on this position the amino acid Tryptophan. Therefore, the mutation on this position is highly conserved and a mutation there will cause probably huge structural and functional changes for the protein.

Mutation in the multiple alignment

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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 the protein.

JPred:
...HHHHCCCCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHCCC...
PsiPred:
...HHHCCCCCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCC...

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.

Mutation at position 485

Mutation at position 485 - detailed view

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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 Arginine which is the real mutation in this case. SNAP has a result that the exhange from Tryptophan to Arginine at this position is non-neutral with a very high accuracy. This means that this certain mutation on this position cause very likely structural and functional changes of the protein.

Substitution	Prediction	Reliability Index	Expected Accuracy
R	Non-neutral	7	96%

A detailed list of all possible substitutions can be found [here]

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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 mutationposition 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.

In this case, the only substitution that is tolerated is the one to Tryptophan itself. The substitution to Arginine is not-tolerated at this position. This means that this mutation at this position is probably not neutral and will cause probably 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.

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.

Predict Not Tolerated Position Seq Rep Predict Tolerated y v t s r q p n m l k i h g f e d c a 485W 0.97 W

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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 both models predict that the mutation is probably damaging. This means that the mutation is not neutral and will probably destroy the structure and the function of the protein

HumDiv prediction

HumVar prediction

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Structure-based Mutation Analysis

Mapping onto Crystal Structure

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 Cystein

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SCWRL Prediction

picture original aa	picture mutated aa	combined picture
Amino acid Histidine	Amino acid Aspartate	Picture which visualize the mutation

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FoldX Energy Comparison

Original total energy	Total energy for the mutated protein	Strongest energy changes within the mutated protein
-154.17	-149.08	-

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