Task 6: MSUD - Sequence-based mutation analysis

Sequence-based mutation analysis

Task description

For this Task the group that reviewed our page last week was to chose 10 SNP's on which we can work on. We assume we don't know which of the SNP's cause MSUP or affects the protein structure or function. As we haven't received any message from the group which reviewed our page last week by thursday, we've decided to chose the 10 SNP's by ourselves: L17F, M82I,Q125E, I213T, C258Y, T310R, A328T, I361V, R429H, N404S

Comparison of wild type AA and mutant AA

Physiological properties<ref>http://en.wikipedia.org/wiki/Amino_acid</ref> and predicted secondary structure element (reprof with pssm)

Position	wt-AA	wt properties	mutant-AA	mutant properties	expected effect on protein	sec-struct-element( reprof )
17	L	-Hydrophobic side chain, non-polar, charge: neutral	F	Hydrophobic side chain, non-polar, charge: neutral	-	L
82	M	Hydrophobic side chain, non-polar, charge: neutral	I	Hydrophobic side chain, non-polar, charge: neutral	-	E
125	Q	polar, charge: neutral	E	polar, charge: negative	+	L
213	I	Hydrophobic side chain, non-polar, charge: neutral	T	polar, charge: neutral	+	H
258	C	polar, charge: neutral	Y	polar, charge: neutral	-	L
310	T	polar, charge: neutral	R	polar, charge: positive	+	H
328	A	non-polar, charge: neutral	T	polar, charge: neutral	-	E
361	I	Hydrophobic side chain, non-polar, charge: neutral	V	non-polar, charge: neutral	-	H
404	N	polar, charge: neutral	S	polar, charge: neutral	-	L
429	R	polar, charge: positive	H	polar, charge: positive( 10% ) negative( 90% )	+	H

Blosum62, PAM1, PAM250 scores

The scores for an aminoacid change in the matrices Blosum and PAM should give another hint, whether the substitution has an effect on the resulting protein or not. The higher the X in BlosumX, the shorter is the evolutionary context of sequences it is calculated for, while in PAMX the opposite is the case.calculated for sequences

Position	wt-AA	mutant-AA	Blosum62-Score<ref>http://www.uky.edu/Classes/BIO/520/BIO520WWW/blosum62.htm</ref> is	PAM1-Score<ref>http://www.icp.ucl.ac.be/~opperd/private/pam1.html</ref>	PAM250-Score<ref>http://www.icp.ucl.ac.be/~opperd/private/pam250.html</ref>
17	L	F	0	13	13
82	M	I	1	5	2
125	Q	E	2	27	7
213	I	T	-2	7	4
258	C	Y	-2	3	4
310	T	R	-1	2	5
328	A	T	-1	32	11
361	I	V	1	33	9
404	N	S	1	20	5
429	R	H	0	10	6

3D-Structure

We used pyMols Mutagenesis Wizard to visualize the individual Mutations. In each image the C-chain of the original residue is coloured green, that of the SNP is coloured pink. Oxygen atoms are coloured red and nitrogen blue, while sulphur is coloured yellow for both residues. The L17F SNP is not displayed because it was not in the pdb file we used (1DTW). The easiest to spot changes that look like the might have an effect are C258Y, T310R and R429H. In case of the C258Y mutation the protein looses a sulphur atom and gains an aromatic ring, while the T310R mutation greatly increases the size of the amino acid. In case of the R429H mutation the protein again gains an aromatic ring.

M82I	Q125E	I213T	C258Y	T310R
A328T	I361V	N404S	R429H

PSI-BLAST - PSSM

the command we used to create the PSSM-file, and the file itsself can be found in the journal

The underlying database is big_db.

Results:

pos	wt-AA	mut-AA	pssm-score	pssm percent wt	pssm percent mut
17	L	F	1	31	9
82	M	I	1	28	6
125	Q	E	-1	97	0
213	I	T	-2	41	2
258	C	Y	-4	29	0
310	T	R	-3	60	0
328	A	T	2	59	12
361	I	V	2	63	13
429	R	H	-1	26	1
404	N	S	2	19	16

Tools

Comparison

Figure 1. Venn-Diagram of the SNPs predicted from Polyphen, SIFT and SNAP that are assumed to have a negative effect on protein functionality.

A visual comparison of the predictions can be seen in Figure 1. For the comparison we only used results from SNAP2. The first observation made when comparing the three predictions is, that the number of SNPs that are predicted to have a negative effect greatly varies between the tools. While PolyPhen predicts seven of the SNPs to be deleterious, SIFT predicts five and SNAP only four to impact protein function. All three methods predicted the SNPs Q125E, T310R and C258Y to have a negative effect, which increses our confidence in the quality of these predictions. PolyPhen and SIFT or SNAP respectively both predicted the L17F and I213T mutation to be deleterious again increasing our confidence in these predictions. None of the tools predicted I361V or N404S to be deleterious and the R429H, M82I and A328T SNPs were only predicted by either SIFT or Polyphen, making them more unlikely.

Figure 2. Venn-Diagram of the SNPs predicted from Polyphen, SIFT, SNAP and Reprof that are assumed to have a negative effect on protein functionality.

When we add the Reprof prediction to this (Figure 2) we observe that the overlap between predictions becomes even larger. There are only Two predictions M82I and A328T from PolyPhen that are not at least supported by one other prediction method. Again only two SNPs (L17F and R429H) are only supported by two methods. While I213T and C258Y are predicted by three and Q125E and T310R even by all four methods.

Polyphen2

SNP	polyphen-score	sensitivity	specificity	description
L17F	0.995	0.68	0.97	PROBABLY DAMAGING
M82I	0.468	0.89	0.90	POSSIBLY DAMAGING
Q125E	0.544	0.88	0.91	POSSIBLY DAMAGING
I213T	0.644	0.87	0.91	POSSIBLY DAMAGING
C258Y	1.000	0.00	1.00	PROBABLY DAMAGING
T310R	1.000	0.00	1.00	PROBABLY DAMAGING
A328T	0.999	0.14	0.99	PROBABLY DAMAGING
I361V	0.386	0.90	0.89	BENIGN
N404S	0.000	1.00	1.00	BENIGN
R429H	0.002	0.99	0.30	BENIGN

Snap/Snap2

SNP	snap-pred	snap-rel	snap-expected-acc	snap2-pred	snap2-rel	snap2-expected-acc
L17F	Neutral	3	78%	Neutral	6	82%
M82I	Neutral	4	85%	Neutral	4	72%
Q125E	Non-neutral	1	63%	Non-neutral	4	71%
I213T	Neutral	3	78%	Non-neutral	6	80%
C258Y	Non-neutral	2	70%	Non-neutral	6	80%
T310R	Non-neutral	1	63%	Non-neutral	6	80%
A328T	Neutral	4	85%	Neutral	1	57%
I361V	Neutral	5	89%	Neutral	8	93%
N404S	Neutral	7	94%	Neutral	9	97%
R429H	Neutral	6	92%	Neutral	4	72%

SIFT

SNP	sift-prediction	sift-score	sift-conservation	sequences represented at pos
L17F	AFFECT PROTEIN FUNCTION	0.00	4.32	1
M82I	be TOLERATED	0.26	3.12	17
Q125E	AFFECT PROTEIN FUNCTION	0.02	3.02	19
I213T	be TOLERATED	0.22	3.02	19
C258Y	AFFECT PROTEIN FUNCTION	0.02	3.02	19
T310R	AFFECT PROTEIN FUNCTION	0.01	3.02	19
A328T	be TOLERATED	1.00	3.02	19
I361V	be TOLERATED	0.06	3.02	19
N404S	be TOLERATED	0.99	3.02	19
R429H	AFFECT PROTEIN FUNCTION	0.03	3.21	15

Discussion

Given that all three prediction methods agree we will assume the mutations Q125E, T310R and C258Y to be deleterious. The actual results in HGMD for our SNPs:

References

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