Difference between revisions of "Gaucher Disease: Task 08 - Sequence-based mutation analysis"
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Revision as of 13:10, 2 September 2013
Mutation Set
<figtable id="sele">
Mutations | |||||
---|---|---|---|---|---|
mRNA | Protein | ||||
Reference | Sequence Position | Codon change | Codon Number | Amino Acid change | One letter code |
rs368786234 | 656 | AGC ⇒ AGA | 77 | Ser ⇒ Arg | S77R |
rs374003673 | 847 | AAT ⇒ AGT | 141 | Asn ⇒ Ser | N141S |
CM880035 | - | CGG ⇒ CAG | 159 | Arg ⇒ Gln | R159Q |
rs374591570 | 1062 | CTC ⇒ TTC | 213 | Leu ⇒ Phe | L213F |
CM992894 | - | GGA ⇒ GAA | 241 | Gly ⇒ Glu | G241E |
rs371083513 | 1470 | GTA ⇒ ATA | 349 | Val ⇒ Ile | V349I |
CM960697 | - | ACG ⇒ ATG | 408 | Thr ⇒ Met | T408M |
CM880036 | - | AAC ⇒ AGC | 409 | Asn ⇒ Ser | N409S |
CM870010 | - | CTG ⇒ CCG | 483 | Leu ⇒ Pro | L483P |
CM057072 | - | AAC ⇒ AGC | 501 | Asn ⇒ Ser | N501S |
</figtable>
Mutation Analysis
In our analysis we looked closer to the amino acid properties and their changing characteristics by mutation. We analysed the structural difference between wild type (WT) and mutation. We also considered their secondary structure and distinguished between helix (H), sheet (E) and loop (C). We also took two different substitution matrices into account, BLOSUM62 and PAM250. Point Accepted Mutation matrix has only positiv integer values as scores and is not symmetric. The score reflects the probability of a amino acid to mutate into another. In contrast the BLOcks SUbstitution Matrix has also negativ integers and is symmetric. A positive score indicates that a substitution occurs more than random. While a score of 0 shows that the substitution occurs randomly, a negative one points to a mutation less frequent than a random mutation. In case one of our selected mutations has the worst possible substitution score for this amino acids we highlighted the score red in <xr id="ana"/>. To consider also evolutionary information we created different PSSM matrices. These position specific scoring matrices are based on alignments. Just as BLOSUM, the PSSM has positive and negative integer values as scores. A positve value shows that the substitution occurs more often than expected. Critical functional residues, like active site residues, have high positive scores. One PSSM was created with a PsiBlast search. The other one is based on an alignment consisting of all mammalian homologous sequences.
<figtable id="ana">
Mutation Analysis | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Changes of Physiochemical Properties | Structural Properties | Conservation | Effect | |||||||||
Mutation | From | To | Pymol Visualization | Secondary Structure | BLOSUM62 score | PAM250 score | PSSM score | PSSM WT frequency | PSSM mutatant frequency | MSA WT frequency | MSA mutant frequency | |
S77R | polar, neutral charge, sulfur-containing | polar, positive, basic | E | -1 | 6 | 1 | 11% | 9% | 64% | 2% | slightly negative | |
N141S | polar, neutral charge, acidic | polar, neutral, sulfur-containing | H | 1 | 5 | 0 | 10% | 7% | 55% | 3% | neutral | |
R159Q | polar, positive charge, basic | polar, neutral, acidic | E | 1 | 5 | -4 | 83% | 0% | 86% | 0% | negative | |
L213F | nonpolar, neutral charge, aliphatic, hydrophobic | nonpolar, neutral, aromatic, hydrophobic | E | 0 | 13 | 3 | 22% | 13% | 100% | 0% | ||
G241E | nonpolar, neutral charge, aliphatic | polar, negative, acidic | C | -2 | 9 | -1 | 10% | 3% | 83% | 0% | ||
V349I | nonpolar, neutral charge, aliphatic, hydrophobic | nonpolar, neutral, aliphatic, hydrophobic | E | 3 | 4 | 0 | 14% | 5% | 97% | 3% | neutral | |
T408M | polar, neutral charge, hydroxyl-containing | nonpolar, neutral, sulfur-containing | H | -1 | 5 | -1 | 4% | 2% | 82% | 0% | ||
N409S | polar, neutral charge, acidic | polar, neutral, sulfur-containing | H | 1 | 5 | 1 | 10% | 9% | 76% | 2% | ||
L483P | nonpolar, neutral charge, aliphatic, hydrophobic | nonpolar, neutral, cyclic | E | -3 | 5 | -3 | 29% | 1% | 100% | 0% | high negative | |
N501S | polar, neutral charge, acidic | polar, neutral, sulfur-containing | E | 1 | 5 | -2 | 87% | 3% | 86% | 1% | negative |
</figtable>
Based on the analysis summed up in <xr id="ana"/> we interpreted our mutations:
S77R : The biggest change happens in the secondary structure. While serine has a short and neutral side chain, arginine shows a much longer positive side chain, that probably causes a clash with the flexible loops of the environment. Additional to the change in its polarity the residue switches from sulfur containing to basic. This could destabilize its secondary strucure, as the parralel located sheet may be not fixed anymore to the sheet of the residue. The PSSM show only no high frequency for the WT as well as the mutant. With scores of -1 and 6, the substitution matrices identifies the point mutation as expectable. We think the only effect comes from the structural change and has a slightly negative effect.
N141S : The mutation causes no change in its charge and polarity. The affected residue is located in a helix on the protein surface which let us assume that no effect may occur (neutral). The substitution matrices as well as the PSSM score confirms us to this opinion, as the scores (1[BLOSUM], 5[PAM] and 0[PSSM]) indicate the mutation as nearly random. Also the PSSM frequencys tells us that the mutation is rare (7% and 3%) and the WT not very distinct (10% and 55%).
R159Q :
L213F :
G241E :
V349I :
T408M :
N409S :
L483P : There seem to be no great changes in the pysichemical properties except the cyclic characteristic of proline. The residue is located at the end of a sheet and may be not important for the structure stabilisation, but it is obvious that the ring clashes with following residues of the adjoining loop which can be severe for the structure. Especially the BLOSUM and PSSM scores (both -3) show an extremly negative effect. The frequency balance between mutation and wild type is very dissimilar (29%>>1%). By looking at the evolutionary information of the homologs, we can see that the WT is present in all sequences. This can give as a hint ti the severeness of the mutation. The reason for the mutant, never showing up, can be that the mutation causes not only a disease but death. All information about this mutation let us identify a high negativ effect.
N501S :
Comparison of different approaches
<figtable id="app">
Summary of different prediction approaches | |||||||
---|---|---|---|---|---|---|---|
Mutation | Analysis of <xr id="ana"/> | SIFT | Polyphen2 (HumDiv) | MutationTaster | SNAP | Conssensus | Validation |
S77R | slightly negative effect | 0.37 | 0.17 | 90% | 2 | dbSNP | |
N141S | no effect | 0.15 | 0.01 | 83% | 5 | dbSNP | |
R159Q | Analysis of <xr id="ana"/> | 0 | 1 | 100% | 7 | HGMD | |
L213F | Analysis of <xr id="ana"/> | 0 | 0.79 | 100% | 0 | HGMD | |
G241E | Analysis of <xr id="ana"/> | 0.01 | 0.89 | 100% | 0 | HGMD | |
V349I | Analysis of <xr id="ana"/> | 0.25 | 0.12 | 100% | 5 | dbSNP | |
T408M | Analysis of <xr id="ana"/> | 0.03 | 0.11 | 60% | 5 | HGMD | |
N409S | Analysis of <xr id="ana"/> | 0.05 | 0.23 | 100% | 0 | HGMD | |
L483P | high negative | 0 | 0.85 | 100% | 1 | HGMD | |
N501S | Analysis of <xr id="ana"/> | 0 | 0.98 | 100% | 0 | HGMD |
</figtable>