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 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>.
| 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 oxigen 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 so 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 chaine 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 comparisson of the accesion number with the OMIM database, we belief that the position is not correct annotated in dbSNP. The position reportet 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 left 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 found here. We created then a multible sequence alignment using ClustalW to calculate the conservation of the SNP positions.
| Mutation | Position | Conservation(JalView) |
|---|---|---|
| S/C | 65 | 10 |
| I/T | 105 | 7 |
| Q/H | 127 | 10 |
| C/Y,S | 282 | 10 |
| R/M | 330 | 7 |
| A/V | 176 | 10 |
| R/S | 6 | 3 |
| T/I | 217 | 10 |
| M/T | 35 | 10 |
SIFT
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 | Cause disease |
|---|---|---|---|---|
| S/C | 65 | AFFECT PROTEIN FUNCTION | 0.00 | yes |
| I/T | 105 | AFFECT PROTEIN FUNCTION | 0.00 | yes |
| Q/H | 127 | TOLERATED | 0.16 | yes |
| C/Y,S | 282 | AFFECT PROTEIN FUNCTION | 0.00 | yes |
| R/M | 330 | TOLERATED | 0.06 | yes |
| A/V | 176 | AFFECT PROTEIN FUNCTION | 0.01 | yes |
| R/S | 6 | AFFECT PROTEIN FUNCTION | 0.01 | yes |
| T/I | 217 | TOLERATED | 1.00 | no |
| M/T | 35 | TOLERATED | 1.00 | no |
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
| Mutation | Position | Prediction | Score | Cause disease |
|---|---|---|---|---|
| S/C | 65 | PROBABLY DAMAGING | 0.997 | yes |
| I/T | 105 | PROBABLY DAMAGING | 0.998 | yes |
| Q/H | 127 | BENIGN | 0.002 | yes |
| C/Y,S | 282 | PROBABLY DAMAGING | 1.000/0.997 | yes |
| R/M | 330 | PROBABLY DAMAGING | 0.948 | yes |
| A/V | 176 | PROBABLY DAMAGING | 0.998 | yes |
| R/S | 6 | PROBABLY DAMAGING | 0.738 | yes |
| T/I | 217 | BENIGN | 0.195 | no |
| M/T | 35 | PROBABLY DAMAGING | 0.989 | no |
References
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