Sequence-based mutation analysis

SNP's

HUMAN_HFE with SNPs from above, HGMD:green, dbSNP:red and both:blue

Because the HFE-Gen has no annotated functional site, we can just adress the biochemical changes for each SNP. A change in functionality or stability can not be described.
To compare the biochemical properties of the amino acid's, we used the very convenient function of wolfram alpha and the Wikipedia entry about amino acids <ref>http://en.wikipedia.org/wiki/Amino_acid</ref>. All SNPs are visualized at the following picture of the HFE_HUMAN UniProt sequence.

Mutation	Position	Database	Blosum62	PAM1	Pam250	Physicochemical changes
S/C	65	HGMD/dbSNP	-1	5	3	There is no change in size and charge, just the polarity changes because an oxygen atom is replaced by a sulfur atom.
I/T	105	HGMD/dbSNP	-1	11	6	An OH-group is replaced by an ethyl-group which leads to a small change in size and a large change in hydrophobicity.
Q/H	127	HGMD/dbSNP	0	20	7	Because of the aromatic ring, the flexibility is reduced with a histidine at this position. The polarity and hydrophobicity remain the same.
C/Y,S	282	HGMD/dbSNP	-2/-1	3/11	3/7	In both cases, the hydrophobicity is changed. Cysteine is a nonpolar whereas Serine and Tyrosine are polar amino acids.
R/M	330	HGMD	-1	1	1	The charge of the side chain changes from positive to neutral and the size is changing but this should not have such a strong impact like the different charge.
A/V	176	HGMD	0	13	9	Alanin and Valine are pretty similar in there properties, just the size changes and the hydrophobicity decreases, but both are water soluble.
R/S	6	HGMD	-1	11	6	The polarity is changing which has an impact on the surface on the water solubility of the protein. Also the size of the side chain changes.
T/I	217	dbSNP	-1	7	4	An ethyl-group is replaced by an OH-group which leads so a small change in size and a large change in hydrophobicity.
M/T	35	dbSNP	-1	6	5	The polarity is changing which has an impact on the surface on the water solubility of the protein.
R/M	58	dbSNP	-1	1	1	The charge of the side chain changes from positive to neutral and the size is changing but this should not have such a strong impact like the different charge.

A change in polarity is just important for residues at the surface of the protein, because with a change in polarity, the hydrophobicity changes and so the water solubility decreases/increases.

Remark:
The reference sequence from UniProt has at position 58 a F, the SNP at this position is annotated as a R to M change. After a comparison of the accession number with the OMIM database, we believe that the position is not correct annotated in dbSNP. The position reported in OMIM is 330, therefore we treated these SNP's as the same and ignored the position 58 SNP. But because we had no SNP's occuring only in dbSNP and HGMD is annotating only disease realated SNP's, we decided to not replace this SNP by another one.

SNP analysis by Hand

First we searched in a non redundant database for homologoues sequences in mammalians. The result list can be found here. We created then a multiple sequence alignment using COBALT to calculate the conservation of the SNP positions.

Mutation	Position	Conservation(JalView)	Secondary Structure (UniProt)
S/C	65	7	Beta-Strand
I/T	105	7	Helix
Q/H	127	2	---
C/Y,S	282	11	Beta-Strand
R/M	330	6	---
A/V	176	7	Helix
R/S	6	0	---
T/I	217	0	---
M/T	35	0	Beta-Strand

We removed all nonsensical sequences like short ones and sequences with an obvious large insertion or deletion.

Psi-Blast

• command line: blastpgp -i hfe.fasta -d /data/blast/nr/nr -e 10E-6 -j 5 -Qpsiblast.mat -o psiblast.out

Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
          A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V   A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
   6 R   -2  2 -3 -4 -2 -2 -3 -4 -3  1  4 -2  1  0 -4 -3 -2 -3 -2  1    0  17   0   0   0   0   0   0   0   0  72   0   0   3   0   0   0   0   0   8  0.51 0.24
  35 M    0 -2 -1 -2 -2 -1 -2 -2 -2 -1 -1 -2  3 -2 -2  0  5 -3 -2  0    3   0   1   0   0   0   0   0   0   0   0   0  17   0   0   0  78   0   0   1  0.66 0.25
  65 S    1 -2  0 -1 -2 -1 -1 -1 -2 -3 -3 -1 -2 -3 -2  5  1 -4 -3 -2    1   0   0   0   0   0   0   0   0   0   0   0   0   0   0  99   0   0   0   0  0.80 0.25
 105 I    1 -3 -4 -4 -2 -3 -3 -3 -3  2  4 -3  1 -1 -3 -2 -2 -3 -2  1   19   0   0   0   0   0   0   0   0  17  64   0   0   0   0   0   0   0   0   1  0.50 0.25
 127 Q   -1 -2 -1 -2 -3  2 -1  6 -2 -4 -4 -2 -3 -4 -3 -1 -2 -3 -4 -4    0   0   0   0   0  17   2  81   0   0   0   0   0   0   0   0   0   0   0   0  1.06 0.25
 176 A    5 -2 -2 -2 -1 -1 -1  0 -2 -2 -2 -1 -2 -3 -2  0 -1 -3 -3 -1   96   0   0   0   0   0   4   1   0   0   0   0   0   0   0   0   0   0   0   0  0.72 0.25
 217 T    0 -2  0 -1 -2 -1 -1 -1 -2 -3 -3 -1 -2 -3 -1  5  3 -4 -2 -2    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  83  17   0   0   0  0.70 0.25
 282 C   -1 -4 -4 -4 10 -4 -5 -3 -4 -2 -2 -4 -2 -3 -4 -2 -2 -3 -3 -2    0   0   0   0 100   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  1.89 0.25
 330 R   -3  2 -4 -4  3 -2 -3 -3 -3 -3 -3 -2 -2  0 -4 -3 -3 11  1 -3    0  19   0   0  10   0   0   0   0   0   0   0   0   0   0   0   0  70   1   0  1.90 0.26

SIFT

SIFT (Sorting Intolerant From Tolerant) uses the knowledge, that important amino acids are more conservered and therefore less mutable than non important amino acids. It predicts if the mutation affects the protein function or not. SIFT was created in 2003 by Ng P. and Henikoff S.

• Server: http://sift.jcvi.org/www/SIFT_seq_submit2.html

The prediction of SIFT is marked with a low confidence warning because, the sequences used for the prediction were not diverse enough.

Mutation	Position	Prediction	Score
S/C	65	AFFECT PROTEIN FUNCTION	0.00
I/T	105	AFFECT PROTEIN FUNCTION	0.00
Q/H	127	TOLERATED	0.16
C/Y,S	282	AFFECT PROTEIN FUNCTION	0.00
R/M	330	TOLERATED	0.06
A/V	176	AFFECT PROTEIN FUNCTION	0.01
R/S	6	AFFECT PROTEIN FUNCTION	0.01
T/I	217	TOLERATED	1.00
M/T	35	TOLERATED	1.00

The complete prediction for each position and amino acid can be found here

We got three warning messages form SIFT for which we have no explanation at this time.

WARNING: Original amino acid H at position 31 is not allowed by the prediction. 
WARNING: Original amino acid S at position 45 is not allowed by the prediction. 
WARNING: Original amino acid Y at position 230 is not allowed by the prediction.

PolyPhen-2

PolyPhen-2 (Polymorphism Phenotyping Version 2) uses structure and sequence based features to predict the influence of an single nucleotide mutation at an human protein. PolyPhen-2 was established by Adzhubei et al. in 2010.

• Server: http://genetics.bwh.harvard.edu/pph2/

Mutation	Position	Prediction	Score
S/C	65	PROBABLY DAMAGING	0.997
I/T	105	PROBABLY DAMAGING	0.998
Q/H	127	BENIGN	0.002
C/Y,S	282	PROBABLY DAMAGING	1.000/0.997
R/M	330	PROBABLY DAMAGING	0.948
A/V	176	PROBABLY DAMAGING	0.998
R/S	6	PROBABLY DAMAGING	0.738
T/I	217	BENIGN	0.195
M/T	35	PROBABLY DAMAGING	0.989

SNAP

SNAP (screening for non acceptable polymorphisms) uses an neural network approach to predict the functional effects of non-synonymous SNPs. It was developed by Rost B. and Bromberg Y. in 2007. It is installed locally at our VM and therefore useable with the command line.

• command line: snapfun -i hfe.fasta -m muta.txt -o snap.out

Mutation	Position	Prediction	Reliability Index	Expected Accuracy
S/C	65	Non-neutral	3	78%
I/T	105	Non-neutral	3	78%
Q/H	127	Non-neutral	2	70%
C/Y,S	282	Non-neutral	6/5	93%/87%
R/M	330	Neutral	2	69%
A/V	176	Non-neutral	3	78%
R/S	6	Non-neutral	0	58%
T/I	217	Non-neutral	1	63%
M/T	35	Neutral	1	60%

Discussion

General

We tried to gain as much knowledge of the SNP and therefore combined the information of the mutated amino acids, results from SNAP, SIFT and PolyPhen2 and if the mutation occurs inside an secondary structure element. We decided to to introduce the field HGMD/dbSNP disease with the groundtruth according to HGMD or dbSNP, if that mutation causes the disease. We are going to discuss each SNP one by one and will finally give an conclusion based on our opinion whether the mutation is disease causing or not. This statement will be compared against the groundtruth from HGMD/dbSNP.

Mutation 1 [R6S]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
R/S	6	Affect Function	Affect Function	Affect Function	---	yes

Change of the nonpolar, positive Arginine to the polar and neutral Serine indicates some problems, because of the hydrophobocity and the sidechains. According to the substitution matrices occurs that change less often, because is scored with high values. The change is also not inside an secondary structure element, which is good for the protein functionality.

SNAP, SIFT and PolyPhen2 are all in common, that this change is non-neutral and will interrupt the protein function. SNAP has an reliability index of 0, which indicates it is very unsure about its prediction but SIFT and PolyPhen2 are both sure, that their prediction is correct. SIFT scores with 0.01, which is very good and PolyPhen2 with an score of 0.75 which is also very good and coinfident.

Because all three methods came to the same results, we are going to trust them and claim that mutation as non-neutral. According to HGMD/dbSNP is that prediction correct.

Mutation 2 [M35T]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
M/T	35	Tolerated	Tolerated	Affect Function	Beta-Strand	no

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 3 [S65C]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
S/C	65	Affect Function	Affect Function	Affect Function	Beta-Strand	yes

Replacing the polar and neutral Serine with the nonpolar and neutral Cysteine does only change the polarity of the sidechain, because a sulfur atom is replaced with an oxygen atom. Therefore this mutation might not even affect the protein function. The scores of the substitution matrices are in the midrange, which means this change occurs from time to time and is nothing very special. Its medium conserved but placed inside and beta-strand which could affect the protein folding.

SNAP, SIFT and PolyPhen2 are again all coinfident, that this mutation is non-neutral. SNAPs prediction is reliable with an coinfidence score of 3, SIFT scores this mutation with 0.00 and is absolutely sure and PolyPhen2 achieves an score of almost 1.0. All assumptions are correct.

Mutation 4 [I105T]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
I/T	105	Affect Function	Affect Function	Affect Function	Helix	yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 5 [Q127H]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
Q/H	127	Affect Function	Tolerated	Tolerated	---	yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 6 [A176V]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
A/V	176	Affect Function	Affect Function	Affect Function	Helix	yes

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 7 [T217I]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
T/I	217	Affect Function	Tolerated	Tolerated	---	no

hier lassen wir uns noch über die mutation aus und was wir so gelernt haben..

Mutation 8 [C282Y,S]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
C/Y,S	282	Affect Function	Affect Function	Affect Function	Beta-Strand	yes

It is the mutation from the nonpolar and neutral Cysteine to the polar and neutral Tyrosine or Serine, therefore a change in the hydrophobocity occurs. Thyrosine also forms a ring and can influence the protein folding. The change occurs inside an beta-strand which is also bad, because changes inside of secondary structure elements might affect the structure of the protein and thus the function.

The substitution matrices values are high for the change of Cysteine to Serine and low for the change to Thyrosine. That indicates, the replacement with Serine is rare and the change to Thyrosine occurs often. According to JalView is the conversation very high.

SNAP, SIFT and PolyPhen2 came to the conclusion, that the change affects the function of the protein. Looking also at the other results, we came to the same conclusion, because all indicates and breakdown of the protein function.

According to HGMD is our assumption correct, the C282Y,S mutation is the most commonly occuring mutation and causes around 90% of all hemochromatosis cases.

Mutation 9 [R330M]

Mutation	Position	SNAP	SIFT	PolyPhen2	Secondary Structure	HGMD/dbSNP disease
R/M	330	Tolerated	Tolerated	Affect Function	---	yes

The mutation of the nonpolar and positive Arginine to the nonpolar and neutral Methionine is bad because of the change of the polarity. This introduces instantly problems with the hydrophobocity and protein folding. But the mutation is not inside and secondary structure and good conserved, so this change might not affect the protein function. According to the substituion matrices is that change scored with very low values and thus should happen more often and could not be that deleterious.

That statement is also supported by the results of SNAP and SIFT; both define the mutation as neutral. SNAP is not very sure about its prediction with a reliability index score of only 2 but an accurancy of 69%. SIFT is also not sure with score of 0.06 but still states as neutral. However PolyPhen2 lists that mutation clearly as non-neutral with an score of ~0.95.

Although two of our three methods call this mutation neutral, we decided it has to be non-neutral. Because SIFT ans SNAP are both very unsure about their prediction but the PolyPhen2 prediction has an score of almost 1.0, we are going to believe more into PolyPhen2. This decision is also supported by the facts, that a change of polarity inside and protein must introduce problems. Therefore we call that mutation as non-neutral and protein-function affecting.

References

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