Rs61731240

General Information

SNP-id	rs61731240
Codon	179
Mutation Codon	His -> Asp
Mutation Triplet	CAT -> GAT

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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 concluded the possible effect that the mutation could have on the protein.

His	Asp	consequences
aromatic, positive charged, polar, hydrophilic	negative charged, small, polar, hydrophilic	On the one side, both amino acids are polar, but on the other side, His is positively charged, while Asp is negatively charged, which is an essential difference between these both amino acids. Therefore it is very likely, that this change causes big changes in the structure of the protein and the protein therefore will probably not work any longer. Furthermore, the structure of the two amino acids is very different, because of the aromatic ring of His.

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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 Aspartate looks very different to Histidine. Histidine has an aromatical ring. Contrary, Aspartate is smaller and forks at the end of the rest. Furthermore, it is also orientated in a completly different direction. This shows that the amino acids have huge structural differences which will probably cause dramatical effects on protein structure and function.

picture original aa	picture mutated aa	combined picture
Amino acid Histidine	Amino acid Aspartate	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 Histidine to Aspartic acid has very low values that are nearer to the values for the rarest subsitution for PAM1 and PAM250. Contrary, for BLOSOUM62 the value for the amino acid subsitution Histidine to Aspartic acid is average. This means the most frequent subsitution value is almost as far as the rarerest subsitution from the underlying value. The difference between the two PAMs can be ascribed to the different preparations of these two kind of substitutions matrices. The difference between the two PAMs and BLOSUM62 can be ascribed to the different preparations of these two kinds of substitutions matrices. The PAM-matrices are evolutionary models whereas BLOSUM is based on protein families. Therefore probably this mutation is evolutionary not that unlikely whereas within a protein family it is more unusual. Therefore, according to PAM1 and PAM250 a mutation at this position will almost certainly cause structural changes which can affect functional changes. The value from BLOSSUM62 is not realy significant and therefore we are not able to determine effects on 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
3	20 (Gln)	0 (Ile, Met)	4	7 (Gln)	2 (Ala, Cys, Gly, Ile, Leu, Met, Phe, Thr, Trp, Val)	-1	2 (Tyr)	-3 (Cys, Ile, Leu, Val)

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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 Histidine to Aspartic acid at this position is very low and near the value for the rarest substitution. This means that this substitution at this position is likely very uncommon which indicates that this substitution has bad effects as 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	9	-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 colored column. Here we can see, that all the other mammalians have the amino acid Histidine on this position. Therefore, the mutation on this position is highly conserved and a mutation there will cause probably huge structural and functional changes in 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 and the protein does not posses any disordered regions. The change of the coil will probably only take places between two secondary structure elements which will not change.

JPred:
...EEEECCCCCEEEEEECCCCCCCHHHHHHHHHHHHHHCCCEEEEEEECCCCCCCCC...
PsiPred:
...EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCCEEEEEECCCCCCCEEC...

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 agreed with the position of the predicted secondary structure and is within a coil. Like explained above this means a mutation will probably not destroy a secondary structure element which affects no drastical structural change. Otherwise it can cause a change of the position of the two nearest secondary structure element which can has a functional loose as a consequence. We think that a structural change is unlikely, because it is not within a secondary structure element and will therefore not cause extrem changes.

Mutation at position 179

Mutation at position 179 - 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 Aspartic acid which is the real mutation in this case. SNAP has a result that the exhange from Histidine to Aspartiy acid 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
D	Non-neutral	6	93%

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 Histidine itself. The substitution to Aspartic acid 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
	m	w	f	c	i	y	v	l	t	a	s	p	e	r	n	d	k	g	Q	179H	0.99	H

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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 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.