Difference between revisions of "Gaucher Disease: Task 08 - Sequence-based mutation analysis"

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(Mutation Analysis)
(Mutation Analysis)
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==Mutation Analysis==
 
==Mutation Analysis==
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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. '''P'''oint '''A'''ccepted '''M'''utation 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 '''BLO'''cks '''SU'''bstitution '''M'''atrix 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"/>.
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Getting a bit closer to evolution you will have to create a PSSM (position specific scoring matrix) for your protein sequence using PSI-BLAST (5 iterations). How conserved are the WT residues in your mutant positions? How is the frequency of occurrence (conservation) for the mutant residue type? Anything interesting?
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And another step close to evolution: Identify all mammalian homologous sequences. Create a multiple sequence alignment for them with a method of your choice. Using this you can now calculate conservation for WT and mutant residues again. Compare this to the matrix- and PSSM-derived results.
 
<figtable id="ana">
 
<figtable id="ana">
 
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Revision as of 09:59, 31 August 2013

LabJournal

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
Information about 10 randomly selected mutations for glucocerebrosidase taken from HGMD (CM...) and dbSNP (rs...).

</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"/>.


Getting a bit closer to evolution you will have to create a PSSM (position specific scoring matrix) for your protein sequence using PSI-BLAST (5 iterations). How conserved are the WT residues in your mutant positions? How is the frequency of occurrence (conservation) for the mutant residue type? Anything interesting? And another step close to evolution: Identify all mammalian homologous sequences. Create a multiple sequence alignment for them with a method of your choice. Using this you can now calculate conservation for WT and mutant residues again. Compare this to the matrix- and PSSM-derived results. <figtable id="ana">

Mutation Analysis
Changes of Physiochemical Properties Structural Properties Conservation
Mutation From To Pymol Visualization Secondary Structure BLOSUM62 score BLOSUM score
S77R polar, neutral charge, sulfur-containing polar, positive, basic
Mutation of serine (blue) to arginine (orange) on position 77.
E -1 6
N141S polar, neutral charge, acidic polar, neutral, sulfur-containing
Mutation of asparagine (blue) to serine (orange) on position 141.
H 1 5
R159Q polar, positive charge, basic polar, neutral, acidic
Mutation of arginine (blue) to glutamine (orange) on position 159.
E 1 5
L213F nonpolar, neutral charge, aliphatic, hydrophobic nonpolar, neutral, aromatic, hydrophobic
Mutation of leucine (blue) to phenylalanine (orange) on position 213.
E 0 13
G241E nonpolar, neutral charge, aliphatic polar, negative, acidic
Mutation of glycine (blue) to glutamic acid (orange) on position 241.
C -2 9
V349I nonpolar, neutral charge, aliphatic, hydrophobic nonpolar, neutral, aliphatic, hydrophobic
Mutation of valine (blue) to isoleucine (orange) on position 349.
E 3 4
T408M polar, neutral charge, hydroxyl-containing nonpolar, neutral, sulfur-containing
Mutation of threonine (blue) to methionine (orange) on position 408.
H -1 5
N409S polar, neutral charge, acidic polar, neutral, sulfur-containing
Mutation of asparagine (blue) to serine (orange) on position 409.
H 1 5
L483P nonpolar, neutral charge, aliphatic, hydrophobic nonpolar, neutral, cyclic
Mutation of serine (blue) to arginine (orange) on position 483
E -3 5
N501S polar, neutral charge, acidic polar, neutral, sulfur-containing
Mutation of asparagine (blue) to serine (orange) on position 501.
E 1 5
Analysis of the chosen mutations of <xr id="sele"/> in the field of their properties, secondary structure and conservation. The secondary structure can be classified as helix (H), sheet (E) and loop (C). In case a mutations is the worst possible subsitution for this amino acid, the substitution matrix score is coloured red.

</figtable>

Substitution and Phenotype

blossom

PSSM

Multiple Sequence Alignment

Comparison of different approaches

SIFT, Polyphen, Mutationtaster, SNAP -> one table

References

http://en.wikipedia.org/wiki/Amino_acid

hgmd

[GENE%20AND%20%22human%22[ORGN]%20AND%20%22snp%22[SNP_CLASS]%20&cmd=DetailsSearch dbSNP]