Sequence-based mutation analysis

SNP's

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.

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

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

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

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

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

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

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.