Rs61747114

General Information

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

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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.

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.

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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 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 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 aa	picture mutated aa	combined picture
Amino acid Leucine	Amino acid Phenylalanine	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 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 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 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)

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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 aa	most frequent substitution	rarest substitution
-3	3	-5

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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 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.

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 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 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 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 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 248

Mutation at position 248 - 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 Phenylalanine which is the real mutation in this case. SNAP has 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]

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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, there are seven substitutions that are tolerated: Tyrosine, Isoleucine, Aspartic acid, Valine, Phenylalanine, Methionine and Leucine. The substitution to Phenyalanine 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.

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
							c	w	p	e	n	k	g	r	s	t	h	a	Q	248L	1.00	Y	I	D	V	F	M	L

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PolyPhen2 Prediction

Finally, we also regarded the PolyPhen2 prediction for this muation. This prediction visualizes how 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 agreed 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.

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

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 the picture, the mutation is located within a loop and far away from the functional residues. Therefore, we do not know in which way this mutation affects the global structure of the protein.

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