Sequence-based mutation analysis HEXA

Mutations

SNP-id	codon number	mutation codon	mutation triplet
rs4777505	29	Asn -> Ser	AAC -> AGC
rs121907979	39	Leu -> Arg	CTT -> CGT
rs61731240	179	His -> Asp	CAT -> GAT
rs121907974	211	Phe -> Ser	TTC -> TCC
rs61747114	248	Leu -> Phe	CTT -> TTT
rs1054374	293	Ser -> Ile	AGT -> ATT
rs121907967	329	Trp -> TER	TGG -> TAG
rs1800430	399	Asn -> Asp	AAC -> GAC
rs121907982	436	Ile -> Val	ATA -> GTA
rs121907968	485	Trp -> Arg	gTGG -> CGG

Analysis of the mutations

We created for each mutation an extra page. The summary of the analysis can be seen in the Summary Section.

[rs4777505: Asn -> Ser]

[rs121907979: Leu -> Arg]

[rs61731240: His -> Asp]

[rs121907974: Phe -> Ser]

[rs61747114: Leu -> Phe]

[rs1054374: Ser -> Ile]

[rs121907967: Trp -> TER]

[rs1800430: Asn -> Asp]

[rs121907982: Ile -> Val]

[rs121907968: Trp -> Arg]

rs4777505: Asn -> Ser

pysicochemical properities

Asn	Ser	consequences
polar, small, hydrophilic, negatively charged	polar, tiny, hydrophilic, neutral	Both amino acids are polar and hydrophilic. Ser is tiny, Asn therefore is a small amino acid. The biggest difference between these two amino acid is, that Asn is negatively charged and Ser is neutral. But this is not that big difference and therefore we suggest, that this mutation do not delete the structure and function of the protein.

Visualisation of the mutation

picture original aa	picture mutated aa	combined picture
Amino acid Asparagine	Amino acid Serine	Picture which visualize the mutation

Subsitution Matrices values

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
20	36 (Asp)	0 (Cys, Met)	5	7 (Asp)	2 (Cys, Leu, Phe, Trp)	1	1 (Asp, His, Ser)	-4 (Trp)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

JPred:
CCHHHHHHHHHHHHHCCCCCCCEEEEEEEEEECCCEEEEECCCEEEEECCCCCCC...
PsiPred:
CHHHHHHHHHHHHHHHCCCCCCCCCCCCEEEECCCEEEEECCCEEEEECCCCCCC...

Comparison with the real structure:

Mutation at position 29

Mutation at position 29 - detailed view

SNAP Prediction

Substitution	Prediction	Reliability Index	Expected Accuracy
S	Neutral	8	96%

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.

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 w 29N 0.78 c f M H i Y p V g L R N d Q T A S K E

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs121907979: Leu -> Arg

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	Arg	consequences
aliphatic, hydrophobic, neutral	positive charged, polar, hydrophilic	Leucine is smaller and without a positive charge. Therefore, Arg is too big for the position of Leu, therefore, the change of Leu with Arg has to cause changes in the 3D structure of the protein. Furthermore, Leu is a hydrophobic amino acid, whereas Arg is hydrophilic. This is the complete contrary and therefore we suggest, that the protein will not function any longer.

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 has a longer chain than Leucine. Contrary Leucine comes to a fork at the end of its rest. This shows that the amino acids have some structural differences that are not drastical, but can be essential.

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

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 Arginine has very low values that are near the values for the rarest subsitution for PAM1 and PAM250. Furthermore, the value for the most frequent substitution differs also a lot from the value for this certain mutation for both PAMs. Contrary for BLOSUM62 the value for the amino acid subsitution Leucine to Arginine is average. This means the most frequent subsitution value is as far as the rarerest subsitution from the the underlying value. 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 famalies. Therefore probably this mutation is evolutionary not that unlikely whereas within a protein family it is unusual.

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
1	22 (Ile)	0 (Asp, Cys)	4	20 (Met)	2 (Cys)	-2	0 (Phe)	-4 (Asp, Gly)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

JPred:
CCHHHHHHHHHHHHHCCCCCCCEEEEEEEEEECCCEEEEECCCEEEEECCCCCCC...
PsiPred:
CHHHHHHHHHHHHHHHCCCCCCCCCCCCEEEECCCEEEEECCCEEEEECCCCCCCC...

Comparison with the real structure:

Mutation at position 39

Mutation at position 39 - detailed view

SNAP Prediction

Substitution	Prediction	Reliability Index	Expected Accuracy
R	Non-neutral	5	87%

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.

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 h d w q p n e c r g s k y t a f m 39L 0.79 V I L

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs61731240: His -> Asp

pysicochemical properities

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 the His.

Visualisation of the mutation

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

Subsitution Matrices values

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)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

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

Comparison with the real structure:

Mutation at position 179

Mutation at position 179 - detailed view

SNAP Prediction

Substitution	Prediction	Reliability Index	Expected Accuracy
D	Non-neutral	6	93%

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.

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

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs121907974: Phe -> Ser

pysicochemical properities

Phe	Ser	consequences
polar, tiny, hydrophilic, neutral	aliphatic, hydrophobic, neutral	Ile is much bigger than Ser and also is branched, because it is an aliphatic amino acid. Therefore the structure of both amino acids is really different and Ile is to big for the position where Ser was. Therefore, there has to be a big change in the 3D structure of the protein and the protein probably will loose its function.

Visualisation of the mutation

picture original aa	picture mutated aa	combined picture
Amino acid Phenylalanine	Amino acid Serine	Picture which visualize the mutation

Subsitution Matrices values

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	28 (Tyr)	0 (Asp, Cys, Glu, Lys, Pro, Val)	2	20 (Tyr)	1 (Arg, Asp, Cys, Gln, Glu, Gly, Lys, Pro)	-2	3 (Tyr)	-4 (Pro)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

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

Comparison with the real structure:

Mutation at position 211

Mutation at position 211 - detailed view

SNAP Prediction

Substitution	Prediction	Reliability Index	Expected Accuracy
S	Non-neutral	5	87%

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.

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 w v t s r q p n m l k i h g e d c a 211F 1.00 F

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs61747114: Leu -> Phe

pysicochemical properities

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

picture original aa	picture mutated aa	combined picture
Amino acid Leucine	Amino acid Phenylalanine	Picture which visualize the mutation

Subsitution Matrices values

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)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

JPred:
...HHHHHHHHCCCEEEECCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCC...
PsiPred:
...HHHHHHHHCCCEEEECCCCCHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCH...

Comparison with the real structure:

Mutation at position 248

Mutation at position 248 - detailed view

SNAP Prediction

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.

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

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs1054374: Ser -> Ile

pysicochemical properities

Ser	Ile	consequences
polar, tiny, hydrophilic, neutral	aliphatic, hydrophobic, neutra	Ile is much bigger than Ser and also is branched, because it is an aliphatic amino acid. Therefore the structure of both amino acids is really different and Ile is to big for the position where Ser was. Therefore, there has to be a big change in the 3D structure of the protein and the protein probably will loose its function.

Visualisation of the mutation

picture original aa	picture mutated aa	combined picture
Amino acid Serine	Amino acid Isoleucine	Picture which visualize the mutation

Subsitution Matrices values

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	38 (Thr)	1 (Leu)	5	9 (Ala, Gly, Pro, Thr)	3 (Phe)	-2	1 (Ala, Asn, Thr)	-3 (Trp)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

JPred:
...HHHHHHHHCCCEEEECCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCC...
PsiPred:
...HHHHHHHHCCCEEEECCCCCHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCH...

Comparison with the real structure:

Mutation at position 293

Mutation at position 293 - detailed view

SNAP Prediction

Substitution	Prediction	Reliability Index	Expected Accuracy
I	Neutral	2	69%

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.

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 w g h y d r n f q e k c p 293S 1.00 M l A S V T I

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs121907967: Trp -> TER

pysicochemical properities

Trp	TER	consequences
aromatic, polar, hydrophobic	TER	By this change, the protein is not complete, therefore it is not possible for the protein to fold and to function.

Visualisation of the mutation

picture original aa	picture mutated aa	combined picture
Amino acid Tryptophan		Visualization of the mutated protein

Subsitution Matrices values

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

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

JPred:
...CCCCCCCCCCCCHHHHHHHHHCCCCCCHHHHHHHHHHHHHHHHHHCCCEEEEECC...
PsiPred:
...CCCCCCCCCCCCHHHHHHHHHCCCCCCHHHHHHHHHHHHHHHHHHCCCCEEEECC...

Comparison with the real structure:

Mutation at position 329

Mutation at position 329 - detailed view

SNAP Prediction

No prediction available, because the protein ends here.

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.

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs1800430: Asn -> Asp

pysicochemical properities

Asn	Asp	consequences
polar, small, hydrophilic, negatively charged	polar, small, hydrophilic, negatively charged	Both amino acids have the same properities and therefore we suggest that an exchange of these two amino acids do not destroy the protein structure and function

Visualisation of the mutation

picture original aa	picture mutated aa	combined picture
Amino acid Asparagine	Amino acid Aspartic acid	Picture which visualize the mutation

Subsitution Matrices values

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
36	36 (Asp)	0 (Cys, Met)	7	7 (Asp)	2 (Cys, Leu, Phe, Trp)	1	1 (Asp, His, Ser)	-4 (Trp)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

JPred:
...CCCCCCCEEEEEECCCCCCCCHHHHHHHHHCCCCEEECCCCCCCCCCCCCCCCCC...
PsiPred:
...CCCCCCCCEEEEECCCCCCCHHHHHHHHHHCCCEEEECCCCCCCCCCCCCCCCCC...

Comparison with the real structure:

Mutation at position 399

Mutation at position 399 - detailed view

SNAP Prediction

Substitution	Prediction	Reliability Index	Expected Accuracy
D	Neutral	1	60%

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.

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 w y f c m 399N 0.72 i H v l g P t R D E Q A S K N

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs121907982: Ile -> Val

pysicochemical properities

Ile	Val	consequences
aliphatic, hydrophobic, neutra	aliphatic, hydrophobic, neutral	In this case, the pysicochemical properties are equal. Furthermore, they almost agree in their size. Therefore, we suggest, that there is no big effect on the 3D structure of the protein and therefore, also no big effect on the protein function.

Visualisation of the mutation

picture original aa	picture mutated aa	combined picture
Amino acid Isoleucine	Amino acid Valin	Picture which visualize the mutation

Subsitution Matrices values

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
33	33 (Val)	0 (Gly, Pro, Trp)	9	9 (Val)	1 (Trp)	3	3 (Val)	-4 (Gly)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

JPred:
...HHHHHHHHCCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEE...
PsiPred:
...HHHHHHHHCCCEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHCCCCCE...

Comparison with the real structure:

Mutation at position 436

Mutation at position 436 - detailed view

SNAP Prediction

Substitution	Prediction	Reliability Index	Expected Accuracy
V	Neutral	0	53%

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.

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 w c 436I 1.00 f Y H M p I V g L n T Q D R A S E K

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

rs121907968: Trp -> Arg

pysicochemical properities

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.

Visualisation of the mutation

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

Subsitution Matrices values

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)

Conservation analysis with multiple alignments

Mutation in the multiple alignment

Secondary structure mutation analysis

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

Comparison with the real structure:

Mutation at position 485

Mutation at position 485 - detailed view

SNAP Prediction

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

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.

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

Polyphen2 Prediction

HumDiv prediction

HumVar prediction

Summary page

method	mutations
	Asn -> Ser	Leu -> Arg	His -> Asp	Phe -> Ser	Leu -> Phe	Ser -> Ile	Trp -> TER	Asn -> Asp	Ile -> Val	Trp -> Arg
pysiochemical properitites	neutral	non-neutral	non-neutral	non-neutral	neutral	non-neutral	non-neutral	neutral	neutral	non-neutral
visuale analysis	todo	non-neutral	non-neutral	non-neutral	todo	neutral	non-neutral	todo	neutral	non-neutral
PAM1	neutral	non-neutral	non-neutral	non-neutral	no statement	non-neutral	no information	neutral	neutral	neutral
PAM250	neutral	non-neutral	no statement	non-neutral	no statement	no statement	no information	neutral	neutral	neutral
BLOSUM62	neutral	no statement	no statement	non-neutral	neutral	non-neutral	no information	neutral	neutral	non-neutral
multiple alignment	neutral	neutral	non-neutral	non-neutral	non-neutral	neutral	neutral	neutral	neutral	non-neutral
analysis with Jpred	non-neutral	non-neutral	neutral	neutral	neutral	neutral	neutral	neutral	neutral	neutral
analysis with PsiPred	non-neutral	non-neutral	neutral	neutral	neutral	neutral	neutral	non-neutral	neutral	neutral
analysis with real structure	non-neutral	no statement	neutral	neutral	non-neutral	neutral	non-neutral	no statement	non-neutral	non-neutral
SNAP Prediction	neutral	non-neutral	non-neutral	non-neutral	neutral	neutral	no information	neutral	neutral	non-neutral
SIFT Prediction	neutral	non-neutral	non-neutral	non-neutral	neutral	neutral	no information	neutral	neutral	non-neutral
PolyPhen2 Prediction	neutral	non-neutral	non-neutral	non-neutral	neutral	neutral	no information	neutral	neutral	non-neutral